Methods of controlling cell fate and consequences for disease

ABSTRACT

Provided herein are methods for performing cellular reprogramming that include treatment of somatic cells with an inhibitor of CAF-1, Sumo2, Nutd21, or combinations thereof prior to or during a reprogramming procedure. Such inhibitors can improve both the speed and efficiency of cellular reprogramming. Inhibitors of the CAF-1 complex can also be used in the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Divisional of U.S. patent application Ser. No.15/443,632 filed Feb. 27, 2017, which claims benefit under 35 U.S.C. §120 and is a Continuation of International PCT Application No.PCT/US2015/046903 filed Aug. 26, 2015, which claims benefit under 35U.S.C. § 119(e) of the U.S. Provisional Application No. 62/041,960 filedAug. 26, 2014, and U.S. Provisional Application No. 62/041,968 filedAug. 26, 2014, the contents of which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in it entirety. Said ASCII copy, created on Sep. 22, 2015, isname 030258-085430-PCT_SL.txt and is 5,639 bytes in size.

FIELD OF THE INVENTION

The field of the invention relates to methods and compositions forenhancing cellular reprogramming and for modifying cell fate for thetreatment of disease.

BACKGROUND

Mammalian development is a unidirectional process that depends on theproper establishment and maintenance of lineage-specific transcriptionalprograms to generate a multitude of differentiated cell types (1-3).Genome-wide analyses of gene expression, chromatin structure andassociated modifications during distinct stages of development andacross different somatic cell types support the notion that cellidentity is maintained by stable and conserved chromatin pathways (4).However, the regulators and mechanisms responsible for preserving thesecellular states remain poorly understood.

Ectopic expression of transcription factors is sufficient to overridestable epigenetic programs and hence alter cell fate (5). For example,forced expression of pluripotency-related transcription factors insomatic cells yields induced pluripotent stem cells (iPSCs), which aretranscriptionally, epigenomically, and functionally equivalent toembryonic stem cells (ESCs) (6). Similarly, forced expression oflineage-specific transcription factors drives conversion of heterologouscells into cardiac, neuronal, myeloid and other specialized cell types(7). Reprogramming transcription factors such as Oct4 and Sox2 arethought to act as “pioneer factors”, which bind to nucleosomal DNA andgradually remodel local chromatin structure to activate target genes(8). However, the reprogramming process is generally slow andinefficient, coinciding with multiple rounds of cell division andrecruitment of additional cofactors.

SUMMARY

The methods and treatments described herein are based, in part, on thediscovery that inhibitors of the CAF-1 complex, inhibitors of Sumo2, andinhibitors of Nutd21 can be used in a cellular reprogramming protocol,and surprisingly can increase the speed and/or efficiency of cellularreprogramming of somatic cells to induced pluripotent stem cells(iPSCs).

Accordingly, provided herein in one aspect is a method for performingcellular reprogramming, the method comprising: (a) contacting a somaticcell with an inhibitor of the CAF-1 complex, Nudt21, or Sumo2, and (b)subjecting the somatic cell to a reprogramming protocol, therebyreprogramming the somatic cell to an induced pluripotent stem cell(iPSC).

In one embodiment of this aspect and all other aspects described herein,the speed and/or efficiency of cellular reprogramming to iPSCs isincreased in the presence of the inhibitor as compared to the speedand/or efficiency of cellular reprogramming performed in the absence ofthe inhibitor.

In another embodiment of this aspect and all other aspects describedherein, the measure of efficiency of cellular reprogramming comprises anincrease in the total number of reprogrammed cells relative toreprogramming in the absence of a said inhibitor.

In another embodiment of this aspect and all other aspects describedherein, the measure of speed of cellular reprogramming comprises theappearance of reprogrammed cells at an earlier time point than occurswhen reprogramming in the absence of said inhibitor.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor comprises an RNA interference molecule or anantibody.

In another embodiment of this aspect and all other aspects describedherein, the RNA interference molecule comprises an siRNA or an shRNA.

In another embodiment of this aspect and all other aspects describedherein, step (a) is performed before or during step (b).

In another embodiment of this aspect and all other aspects describedherein, the reprogramming of step (b) comprises induction ofOct-4/Klf4/Sox-2/c-Myc (OKSM) expression.

In another embodiment of this aspect and all other aspects describedherein, the reprogramming step does not comprise forced expression ofc-Myc.

In another embodiment of this aspect and all other aspects describedherein, the somatic cell comprises a fibroblast.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor of the CAF-1 complex inhibits the Chaf1a and/orChaf1b subunit of said complex.

Another aspect provided herein relates to a method of inducingdifferentiation of a cancer cell or cancer stem cell in vivo, the methodcomprising: administering an inhibitor of the CAF-1 complex to a subjecthaving, or suspected of having cancer, thereby inducing differentiationof the cancer cell or cancer stem cell in vivo.

In one embodiment of this aspect and all other aspects provided herein,the cancer comprises leukemia.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor comprises an RNA interference molecule or anantibody.

In another embodiment of this aspect and all other aspects describedherein, the RNA interference molecule comprises an siRNA or an shRNA.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor inhibits the Chaf1a and/or Chaf1b subunit of theCAF-1 complex.

Also provided herein in another aspect is a composition comprising: oneor more inhibitors of the CAF-1 complex, Nudt21, or Sumo2 and apharmaceutically acceptable carrier.

In one embodiment of this aspect and all other aspects described herein,the composition comprises inhibitors or any two or all of the CAF-1complex, Nudt21 and Sumo2.

Another aspect provided herein relates to a composition for use incellular reprogramming, the composition comprising an inhibitor of theCAF-1 complex, Nudt21, or Sumo2.

In one embodiment of this aspect and all other aspects provided herein,the composition further comprises a pharmaceutically acceptable carrier.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor increases the total number of reprogrammed cellsrelative to reprogramming in the absence of the inhibitor.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor promotes the appearance of reprogrammed cells atan earlier time point than occurs when cells are reprogrammed in theabsence of the inhibitor.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor comprises an RNA interference molecule or anantibody.

In another embodiment of this aspect and all other aspects describedherein, the RNA interference molecule comprises an siRNA or an shRNA.

In another embodiment of this aspect and all other aspects describedherein, cellular reprogramming comprises induction ofOct-4/Klf4/Sox-2/c-Myc (OKSM) expression.

In another embodiment of this aspect and all other aspects describedherein, cellular reprogramming does not comprise forced expression ofc-Myc.

In another embodiment of this aspect and all other aspects describedherein, cellular reprogramming comprises reprogramming of a fibroblast.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor of the CAF-1 complex inhibits the Chaf1a and/orChaf1b subunit of the complex.

Another aspect provided herein relates to the use of an inhibitor of theCAF-1 complex, Nudt21, or Sumo2 for cellular reprogramming.

In one embodiment of this aspect and all other aspects described herein,the inhibitor increases the speed and/or efficiency of cellularreprogramming.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor comprises an RNA interference molecule or anantibody.

In another embodiment of this aspect and all other aspects describedherein, the RNA interference molecule comprises an siRNA or an shRNA.

In another embodiment of this aspect and all other aspects describedherein, cellular reprogramming comprises induction ofOct-4/Klf4/Sox-2/c-Myc (OKSM) expression.

In another embodiment of this aspect and all other aspects describedherein, the cellular reprogramming does not comprise forced expressionof c-Myc.

In another embodiment of this aspect and all other aspects describedherein, cellular reprogramming comprises reprogramming of a fibroblast.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor of the CAF-1 complex inhibits the Chaf1a and/orChaf1b subunit of the complex.

Another aspect provided herein relates to a composition for use in thetreatment of cancer, the composition comprising an inhibitor of theCAF-1 complex.

In one embodiment of this aspect and all other aspects provided herein,the composition induces the differentiation of a cancer cell or cancerstem cell in vivo when administered to an individual having cancer.

In another embodiment of this aspect and all other aspects describedherein, the composition further comprises a pharmaceutically acceptablecarrier.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor comprises an RNA interference molecule or anantibody.

In another embodiment of this aspect and all other aspects describedherein, the RNA interference molecule comprises an siRNA or an shRNA.

In another embodiment of this aspect and all other aspects describedherein, the cancer comprises a leukemia.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor inhibits the Chaf1a and/or Chaf1b subunit of theCAF-1 complex.

Also provided herein in another aspect is the use of an inhibitor of theCAF-1 complex for the treatment of cancer, the use comprisingadministering said inhibitor of the CAF-1 complex to an individualhaving cancer.

In one embodiment of this aspect and all other aspects provided herein,the administering induces the differentiation of a cancer cell or cancerstem cell, thereby treating said cancer.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor comprises an RNA interference molecule or anantibody.

In another embodiment of this aspect and all other aspects describedherein, the RNA interference molecule comprises an siRNA or an shRNA.

In another embodiment of this aspect and all other aspects describedherein, the cancer comprises a leukemia.

In another embodiment of this aspect and all other aspects describedherein, the inhibitor inhibits the Chaf1a and/or Chaf1b subunit of theCAF-1 complex.

Also provided herein, in another aspect, is a method for performingcellular transdifferentiation, the method comprising: (a) contacting asomatic cell with an inhibitor of the CAF-1 complex, and (b) subjectingthe somatic cell to a transdifferentiation protocol, therebytransdifferentiating the somatic cell to a different cell type.

In one embodiment of this aspect and all other aspects provided herein,the speed and/or efficiency of cellular transdifferentiation isincreased in the presence of the inhibitor as compared to the speedand/or efficiency of cellular reprogramming performed in the absence ofthe inhibitor.

In another embodiment of this aspect and all other aspects providedherein, the measure of efficiency of cellular transdifferentiationcomprises an increase in the total number of transdifferentiated cellsrelative to transdifferentiation in the absence of a said inhibitor.

In another embodiment of this aspect and all other aspects providedherein, the measure of speed of cellular transdifferentiation comprisesthe appearance of transdifferentiated cells at an earlier time pointthan occurs when cells are transdifferentiated in the absence of saidinhibitor.

In another embodiment of this aspect and all other aspects providedherein, the inhibitor comprises an RNA interference molecule or anantibody.

In another embodiment of this aspect and all other aspects providedherein, the RNA interference molecule comprises an siRNA or an shRNA.

In another embodiment of this aspect and all other aspects providedherein, step (a) is performed before or during step (b).

In another embodiment of this aspect and all other aspects providedherein, the transdifferentiation of step (b) comprisestransdifferentiation of a fibroblast to a neuron or transdifferentiationof a B-cell to a macrophage.

In another embodiment of this aspect and all other aspects providedherein, transdifferentiation of a fibroblast to a neuron comprisesoverexpression (e.g., forced expression) of the transcription factorAscl1 in a fibroblast.

In another embodiment of this aspect and all other aspects providedherein, transdifferentiation of a B-cell to a macrophage comprisesoverexpression (e.g., forced expression) of the myeloid transcriptionfactor C/EBPα in a B-cell.

In another embodiment of this aspect and all other aspects providedherein, the inhibitor of the CAF-1 complex inhibits the Chaf1a and/orChaf1b subunit of said complex.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1K. FIG. 1 describes a serial genome-wide shRNA enrichmentscreen during iPSC generation. FIG. 1A, Fluorescence microscopy image ofa primary iPSC colony showing lentiviral tRFP expression and activationof the endogenous Oct4-GFP pluripotency reporter. FIG. 1B, Flowcytometric analysis of tRFP (y axis) and Oct4-GFP (x-axis) expression ofMEFs having undergone reprogramming with gating strategy to purifyOct4-GFP+ cells. FIG. 1C, Timeline of reprogramming experiments andstrategy to collect control and experimental samples for subsequentanalysis of shRNA library representation. FIG. 1D Schematicrepresentation of steps required for every reprogramming and shRNAenrichment cycle. FIG. 1E Overview of serial enrichment screen andvalidation experiments. FIG. 1F Change in shRNA library complexityduring initial screen, i.e., number of unique shRNAs at the start ofrounds 1-3. FIG. 1G, Enrichment of candidate shRNAs during the first 3rounds of reprogramming from screen #1. Lines depict hairpins thatenhanced iPSC formation more than 2-fold. FIG. 1H, Validationexperiments screen #1. FIG. 1I, Gradual loss of shRNA library complexityduring repeat screen; i.e., number of unique shRNAs at the start ofrounds 1-5. FIG. 1J, Heatmap depicting fold-change enrichment of shRNAsduring 5 rounds of reprogramming from screen #2. Note that blue barsrepresent shRNA that were lost whereas red bars represent shRNA thatbecame enriched relative to controls (see text and methods section fordetails). FIG. 1K, Validation experiments screen #2.

FIGS. 2A-2J. FIG. 2 demonstrates that suppression of Nudt21 and Sumo2strongly enhances and accelerates reprogramming. FIG. 2A, Flowcytometric analysis of Oct4-GFP expression in reprogrammable MEFs after8 days or OKSM expression in the presence of hairpins against Fireflyluciferase (control), Sumo2 or Nudt21. tRFP expression depicts cellscarrying lentiviral shRNA vector. FIG. 2B, Quantification of data shownin FIG. 2A; shown is percentage of Oct4-GFP+ cells per total number ofcells using three replicates. FIG. 2C, Alkaline Phosphatase (AP)staining for iPSC colonies derived from reprogrammable MEFs transfectedonce with siRNAs targeting Renilla (control), Nudt21 or Sumo2 duringreprogramming. FIG. 2D, Quantification of data shown in FIG. 2C; dataobtained from three independent replicates. FIGS. 2E-2F, Western blotanalyses for Nudt21 (FIG. 2E) and Sumo2 (FIG. 2F) expression inreprogrammable MEFs infected with respective shRNA vectors and treatedwith doxycycline (dox) for 3 days. FIG. 2G, Expression dynamics ofNudt21 and Sumo2 in MEFs, iPSCs and intermediate stages ofreprogramming. Note that expression of either gene does not changedramatically compared to controls (Thy1, fibroblast marker; Nanog,pluripotency marker). FIG. 2H, Scheme to determine minimal duration ofOKSM expression (in days) required to achieve transgene-independent iPSCcolonies. FIG. 2I, Data obtained from experiments depicted in FIG. 2Gusing shRNAs targeting Firefly luciferase (control), Nudt21 or Sumo2.Shown is quantification of AP+ transgene-independent colonies after 3-10days of dox exposure, followed by at least 4 days of dox withdrawal.FIG. 2J, Expression levels of epigenetic regulators (Dnmt3b, Tet1) andpluripotency-associated genes (EpCAM, Cdh1, Sal14) in indicated samplesat day 6 of OKSM expression or in established iPSCs.

FIGS. 3A-3E. FIG. 3 demonstrates the effect of Nudt21 and Sumo2suppression on defined reprogramming intermediates. FIG. 3A, Overview ofsurface markers and reporter alleles used to distinguish between early,mid and late stages of reprogramming. FIG. 3B, Flow cytometry analysisof indicated markers (SSEA1, EpCAM and Oct4-GFP) at intermediate stagesof reprogramming in the presence of shRNAs targeting Firefly luciferase,Sumo2 or Nudt21. Note that tRFP expression identifies lentivirallytransduced cells. FIG. 3C, Quantification of data shown in FIG. 3B using3 independent replicates. FIG. 3D, AP+ transgene-independent iPSCcolonies obtained upon transfection of reprogrammable MEFs with siRNAstargeting Renilla control, Sumo2 or Nudt21 either once (day 0) or twice(day 0 and day 3) in the presence of dox for 6 days; iPSC colonies werescored after 4 days of dox withdrawal to capture stable iPSC. FIG. 3E,Quantification of data shown in FIG. 3D.

FIGS. 4A-4F. FIG. 4 demonstrates that Nudt21 and Sumo2 suppression actindependently of c-Myc expression and in parallel with small moleculeenhancers of reprogramming. FIG. 4A, Scheme depicting 3-factorreprogrammable MEFs carrying Col1a1-tetOP-OKS-mCherry and Rosa26-M2rtTAalleles to assess effect of c-Myc expression on iPSC formation. FIG. 4B,Generation of AP+ transgene-independent iPSC colonies obtained from3-factor reprogrammable MEF transfected with shRNAs targeting Renillacontrol, Nudt21 or Sumo2 after exposure to dox and small molecules for 9days; data generated from 3 independent replicates. FIG. 4C,Quantification of data shown in FIG. 4B, dox only samples as well asadditional time points. FIG. 4D, Oct4-GFP expression of 3-factorreprogrammable MEFs treated with indicated siRNAs and dox for 9 days,followed by 5 days of dox-independent growth. Note that no Oct4-GFP+iPSCs could be recovered at this time point without Sumo2 or Nudt21suppression. FIG. 4E, Comparison of iPSC formation efficiencies fromreprogrammable (4-factor) MEFs in the presence of either small moleculesor siRNAs targeting Sumo2 and Nudt21. Note the strong effect of Sumo2 orNudt21 suppression relative to well-characterized small molecules oniPSC formation efficiencies. FIG. 4F, Combination treatment ofreprogrammable MEFs with siRNA targeting Sumo2 or Nudt21 and indicatedsmall molecule enhancers of iPSC generation. Note the additive effect ofSumo2 or Nudt21 suppression and small molecule treatment onreprogramming efficiencies.

FIGS. 5A-5F. FIG. 5 demonstrates Generation of iPSCs after as little as36-48 hours of OKSM expression. FIG. 5A, Treatment with ascorbate (AA),Dot11 inhibitor (Dot11i) and Gsk3b inhibitor (Gsk3bi) facilitates therecovery of transgene-independent AP+ iPSC colonies from control andSumo2 or Nudt21 siRNA transfected MEFs after 72 hours of OKSMexpression. FIG. 5B, Quantification of data shown in FIG. 5A using threeindependent replicates. FIG. 5C, Suppression of Sumo2 enables generationof Oct4-GFP+ transgene-independent iPSCs after 38 hours of OKSMexpression whereas suppression of Nudt21 facilitates generation of iPSCsafter 48 hours of OKSM expression in the presence of small molecules.FIG. 5D, Expression of endogenous Oct4, Nanog and Sox2 byimmunofluorescence in iPSCs generated after Sumo2 suppression and OKSMexpression for 38 hours. FIG. 5E, iPSCs shown in FIG. 5C are pluripotentas determined by their potential to differentiate into all threegermlayers in teratomas. FIG. 5F, iPSCs produced with Sumo2 siRNAs andsmall molecules following 38 hours of OKSM expression give rise to coatcolor chimeras.

FIG. 6. FIG. 6 schematically shows the workflow to obtain experimentaland control samples for deep sequencing during initial serial shRNAscreens.

FIG. 7. FIG. 7 shows the results of experiments providing confirmationof reprogramming phenotype with independent shRNAs targeting Sumo2 (leftpanel) or Nudt21 (right panel). Shown are iPSC formation efficienciesafter infecting reprogrammable MEFs with additional shRNAs targetingdifferent seed sequences within Sumo2 and Nudt21 mRNAs. iPSC colonieswere determined after 10 days of dox treatment.

FIG. 8. FIG. 8 shows that shRNAs against Nudt21 and Sumo2 reducetranscript levels of their respective target mRNAs. qPCR analysis forNudt21 or Sumo2 transcripts normalized to Gapdh levels.

FIGS. 9A-9B. FIG. 9 shows that Sumo2 or Nudt21 knockdown do not affectcell proliferation or viability. FIG. 9A, Growth curve of reprogrammableMEFs expressing shRNAs against Firefly, Nudt21 or Sumo2 in the presenceor absence of dox (i.e., OKSM expression). Cell counts were normalizedto the cell number at day 1 (data obtained from 3 replicates). FIG. 9B,Parallel cultures as shown in FIG. 10A were stained for Annexin V(apoptosing cells) and DAPI (dead cells) (data obtained from 3replicates).

FIGS. 10A-10B. FIG. 10 shows the schematic approach and results usingarrayed and multiplexed shRNAmir screening strategies to identifysuppressors of reprogramming. FIG. 10A, Arrayed screen design using acombination of retroviral GFP shRNAmir (pLMN) chromatin library (n=247genes) and Col1A1-OKSM; Rosa26-M2rtTA double transgenic reprogrammableMEFs. Effects of individual shRNAs were tested by counting number ofalkaline phosphatase-positive (AP+), doxycycline (dox) independentiPSCs. FIG. 10B, Compilation of arrayed screen results showing averagereprogramming efficiency ratios of two biological replicates normalizedto Renilla shRNA control.

FIGS. 11A-11C. FIG. 11 shows that CAF-1 suppression acceleratesreprogramming and yields developmentally competent iPSCs. FIG. 11A,Validation of screening result: measurement of Oct4-GFP+ iPSC coloniesobtained after knockdown of either Chaf1a, Chaf1b, or both Chaf1a andChaf1b (pool). FIG. 11B, Expression dynamics of early reprogrammingmarker Epcam (empty circle, solid line) and late reprogramming markerOct4-tomato (full circle dotted line) in CAF-1 depleted and controlcells after four and six days of OKSM expression. FIG. 11C, Alkalinephosphatase-positive, transgene-independent iPSC colonies obtained atday 10, following four or six days of dox treatment in the presence ofindicated Chaf1a or Renilla shRNAs and ascorbate.

FIGS. 12A-12E. FIG. 12 demonstrates that reprogramming phenotype dependson optimal CAF-1 and OKSM dose. FIG. 12A, Comparison of reprogrammingefficiency upon CAF-1 knockdown when using MEFs heterozygous orhomozygous for the Col1a1::tetOP-OKSM and R26-M2rtTA alleles. Shown arealkaline phosphatase-positive, transgene-independent colonies after sixdays of dox treatment and four days of dox withdrawal. FIG. 12B,Quantification of data shown in FIG. 12A. FIG. 12C, Schematic ofCol1a1::tetOP-miR30-Chaf1a knockin allele. FIG. 12D, MEFs carrying theCol1a1::tetOP-miR30-Chaf1a shRNA and R26-M2rtTA alleles were infectedwith constitutive pHAGE (Efla-OKSM) lentiviral expression vector andexposed to either high (2 m/ml, top row) or low (0.2 m/ml, bottom row)doses of dox for indicated time windows before scoring iPSC colonies onday nine by immunostaining for Nanog. FIG. 12E, Quantification of datashown in FIG. 12D.

FIGS. 13A-13I. FIG. 13 shows that CAF-1 suppression enhancesreprogramming in different cell conversion systems. FIG. 13A, Strategyto assess effect of CAF-1 suppression on reprogramming fetalhematopoietic stem and progenitor cells (HSPCs) into iPSCs. FIG. 13B,Flow cytometric analysis for Pecam expression during the reprogrammingof HSPCs into iPSCs upon Chaf1a suppression using two independentshRNAs. FIG. 13C, Quantification of flow cytometry data shown in FIG.13B based on geometric means. FIG. 13D, Schematic oftransdifferentiation assay of MEFs into induced neurons (iNs) upon viralAscl1 expression in the presence of a dox-inducible Chaf1a shRNA allele.FIG. 13E, Representative image of MAP2+ iNs detected after 13 days oftransdifferentiation. Scale bars: 100 um. FIG. 13F, Quantification oftransdifferentiation efficiency, depicted as average number of MAP2+ iNsper 10 frames for each independent experiment (n=5; values aremean+/−S.D; **, unpaired t-test; p=0.0075). FIG. 13G, Assay outline tostudy transdifferentiation of pre-B cells into macrophages using anestradiol-inducible C/EBPα allele, in the presence of CAF-1 or controlshRNAs. FIG. 13H, Representative histograms (n=2) showing activation ofmacrophage markers Cd14 (left) and Mac1 (right) in control (emptyvector; “Null ctrl”) and Chaf1a and Chaf1b knockdown cells after 0, 24and 48 hours of transdifferentiation (estradiol exposure). FIG. 13I,Differences in Cd14 and Mac1 expression levels between Chaf1a/Chaf1bknockdown samples and empty vector control at 0, 24 and 48 hours oftransdifferentiation. Shown are fold-change expression differencesbetween experimental and control samples based on geometric meanscalculated from histogram plots (n=2 independent viral transductions).

FIGS. 14A-14F. FIG. 14 shows that CAF-1 suppression primes pluripotencyloci for transcriptional activation by promoting chromatin accessibilityand Sox2 binding at regulatory elements. FIG. 14A, ATAC-Seq analysis ofCAF-1 and control cells at day three of reprogramming to measure globalchromatin accessibility across ESC-active enhancers (n=14265) andpromoters (n=5513). Shown are merged data for Chaf1a.164 and Chaf1a.2120shRNA infected cells. FIG. 14B, Meta gene analysis of Sox2 ChIP-Seq dataacross all ESC-active promoters and enhancers at day three ofreprogramming. Note that cells infected with the weaker shRNA vector(Chaf1a.2120) exhibit a chromatin state that is in between that of cellsinfected with the Renilla shRNA vector and cells infected with thestronger (Chaf1a.164) shRNA vector. FIG. 14C, Comparison of ATAC-Seq andChIP-Seq data at the Sal11 super-enhancer. Shaded grey bars highlightenhanced Sox2 binding and concomitant accessible chromatin structure atdays three and six of reprogramming upon depletion of CAF-1. Blue tracksdepict ATAC-seq signatures of iPSCs and Sox2 binding patterns in ESCs.FIG. 14D, Analysis of H3K9me3-dependent reprogramming-resistant regions(RRRs) as defined by Matoba et al. 45 in CAF-1 depleted and control MEFs(day 0) and reprogramming intermediates (day 3). Left panel: heatmapshowing the changes in H3K9me3 enrichment over reprogramming resistant(RRR) regions at day 0 and 3 of control (Ren.713) and Chaf1a shRNAknockdown experiments (Chaf1a.164 and Chaf1a.2120). For each RRR (rows),values reflect the averaged H3K9me3 signal between 5 kb intervalsspanning the entire region. Right panel: examples of RRRs where theaverage H3K9me3 signal significantly decreases between day 0 and 3 inthe Chaf1a knockdown cells (p<0.05 for both shRNAs afterBenjamin-Hochberg correction). Boxplots show the distributions ofH3K9me3 signal values for 5 kb intervals spanning across the RRR in eachcondition and time point. FIG. 14E, Correlation of ATAC-Seq data withgene expression data during reprogramming time course. Shown areATAC-seq signals for chromatin regions proximal to genes that becomeupregulated in CAF-1 knockdown intermediates at day 6 by microarray orRNA-seq analysis. ATAC-Seq data are presented for each time point (day0, 3 and 6) and genotype (Renilla and two independent Chaf1a shRNAs).Note that upregulated genes in CAF-1 KD cells at day 6 already show amore accessible chromatin structure at day 3. FIG. 14F, Model:Suppression of CAF-1 during iPSC formation promotes a more accessiblechromatin structure at enhancer elements and increased Sox2 binding toESC-specific targets upon overexpression of reprogramming factors,giving rise to intermediate cells that are more permissive to undergocell fate change.

FIGS. 15A-15B. FIG. 15 demonstrates the validation of hits fromchromatin-focused shRNA screens. FIG. 15A, RT-PCR analysis to confirmknockdown of Chaf1a and Chaf1b expression with Mir30-based vectors fromarrayed screen. FIG. 15B, Western blot analysis to confirm knockdown ofCAF-1 complex using the top-scoring MiR30-based shRNAs from arrayedscreen.

FIGS. 16A-16C. FIG. 16 shows the results of experiments that confirmCAF-1 levels were suppressed during HSPC reprogramming andtransdifferentiation. FIG. 16A, Transgene independence assay during HSPCreprogramming in the presence of CAF-1 or control hairpins. Dox pulseswere given for 3 or 6 days and AP+ colonies were scored 5 days post Doxwithdrawal. FIG. 16B, RT-qPCR analysis for Chaf1a expression to confirmknockdown at day 3 post dox induction (shRNAmir and Ascl1 expression)(n=3; mean+/−S.D.). FIG. 16C, RT-qPCR analysis for Chaf1a and Chaf1bexpression to confirm knockdown in transduced pre-B cells prior toinduction of transdifferentiation.

FIGS. 17A-17C. FIG. 17 shows the results of experiments that demonstratethat CAF-1 promotes accessible chromatin structure at enhancer elements.FIG. 17A, Experimental outline and assays (SONO-seq, ATAC-seq, Sox2ChIP-seq) to dissect effect of CAF-1 suppression on chromatinaccessibility and transcription factor binding, performed at days 3 and6 of reprogramming as well as in established iPSCs. FIG. 17B, Metageneanalysis for ESC-specific promoters and enhancers using ATAC-seqprofiles at day 3 of reprogramming. FIG. 17C, Genome snap shots ofrepresentative ATAC-seq accessibility maps at super-enhancer elements(close to Sox2 and Sal14 loci). Shaded grey bars highlight moreaccessible sites in CAF-1 knockdown samples at days 3 and 6 ofreprogramming compared to Renilla shRNA controls.

DETAILED DESCRIPTION

Provided herein are methods for performing cellular reprogramming thatinclude treatment of somatic cells with an inhibitor of CAF-1, Sumo2,Nutd21, or combinations thereof prior to or during a reprogrammingprocedure. Such inhibitors can improve both the speed and efficiency ofcellular reprogramming. Also described herein are methods in which,rather than reversing cell differentiation, inhibitors of CAF-1 alsosurprisingly promote differentiation, including transdifferentiation andcancer cell differentiation. Thus, CAF-1 inhibition can also be used inthe treatment of cancer.

Definitions

As used herein, the term “cellular reprogramming” refers to a processthat alters or reverses the differentiation state of a differentiatedcell (e.g., a somatic cell). Stated another way, reprogramming refers toa process of driving the differentiation of a cell backwards to a moreundifferentiated or more primitive type of cell. It should be noted thatplacing many primary cells in culture can lead to some loss of fullydifferentiated characteristics. Thus, simply culturing such cellsincluded in the term differentiated cells does not render these cellsnon-differentiated cells (e.g., undifferentiated cells) or pluripotentcells. The transition of a differentiated cell to pluripotency requiresa reprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture. The cell tobe reprogrammed can be either partially or terminally differentiatedprior to reprogramming. In some embodiments, reprogramming encompassesreversion of the differentiation state of a differentiated cell (e.g., asomatic cell) to a pluripotent state or a multipotent state. In someembodiments, reprogramming encompasses complete or partial reversion ofthe differentiation state of a differentiated cell (e.g., a somaticcell) to an undifferentiated cell (e.g., an embryonic-like cell). Theresulting cells are referred to as “reprogrammed cells;” when thereprogrammed cells are pluripotent, they are referred to as “inducedpluripotent stem cells (iPSCs or iPS cells).”

Many varying reprogramming methods are known, and it is reasonable toexpect that the removal of the barriers identified herein will permitincreased speed and/or efficiency for any such method that re-programscells. For the avoidance of doubt, the term “cellular reprogramming inthe absence of the inhibitor” refers to a comparison of, e.g.,reprogramming speed and/or efficiency, between any given reprogrammingregimen and that same regimen performed in the presence of an inhibitorof a factor identified herein as a roadblock to reprogramming. Thus, anygiven reprogramming regimen can serve as the basis for comparison. Thesame is also true with regard to comparisons for the speed or efficiencyof transdifferentiation.

As used herein, the term “speed” when used in reference to cellularreprogramming refers to the appearance of reprogrammed cells at anearlier time point in the presence of an inhibitor or agent as describedherein as compared to the emergence of reprogrammed cells in the absenceof the inhibitor or agent.

As used herein, the term “earlier time point” means that the emergenceof reprogrammed cells in the presence of an inhibitor or agent asdescribed herein occurs at least 6 h earlier, at least 12 h earlier, atleast 18 h earlier, at least 24 h earlier, at least 30 h earlier, atleast 36 h earlier, at least 4 days earlier, at last 5 days earlier, atleast 6 days earlier or more relative to the emergence of reprogrammedcells in the absence of the inhibitor or agent.

As used herein, the term “efficiency of cellular reprogramming” refersto the percentage or number of reprogrammed cells at a given time point.Accordingly, efficiency means that the number of reprogrammed cells inthe presence of an inhibitor or agent is increased at least 10%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 90%, atleast 95%, or even 99% compared to the number of reprogrammed cellsgenerated in the absence of the inhibitor or agent. In some embodiments,the number of reprogrammed cells produced in the presence of aninhibitor or agent is increased by at least 1-fold, at least 2-fold, atleast 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, atleast 100-fold, or more compared to the number of reprogrammed cellsproduced in the absence of the inhibitor or agent.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.,the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,at least about a 20-fold increase, at least about a 50-fold increase, atleast about a 100-fold increase, at least about a 1000-fold increase ormore as compared to a reference level.

The term “pharmaceutically acceptable” refers to compounds andcompositions which may be administered to mammals without unduetoxicity. The term “pharmaceutically acceptable carriers” excludestissue culture medium. Exemplary pharmaceutically acceptable saltsinclude but are not limited to mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like, andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in molecular biologycan be found in The Merck Manual of Diagnosis and Therapy, 19th Edition,published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular CellBiology and Molecular Medicine, published by Blackwell Science Ltd.,1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), MolecularBiology and Biotechnology: a Comprehensive Desk Reference, published byVCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006; Lewin's Genes XI, published byJones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael RichardGreen and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(2012) (ISBN 1936113414); Davis et al., Basic Methods in MolecularBiology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.)Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology(CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), and Current Protocols in Protein Science(CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005 (ISBN0471142735), the contents of which are all incorporated by referenceherein in their entireties.

Cellular Reprogramming

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a hematopoietic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent. Thespecific reprogramming approach or method used to generate pluripotentstem cells from somatic cells is not critical to the claimed invention.Thus, any method that re-programs a somatic cell to the pluripotentphenotype would be appropriate for use in the methods described herein.Non-limiting examples of reprogramming approaches are discussed in thefollowing.

Reprogramming methodologies using defined combinations of transcriptionfactors have been described for generating induced pluripotent stemcells. Yamanaka and Takahashi converted mouse somatic cells to EScell-like cells with expanded developmental potential by the directtransduction of genes encoding Oct4, Sox2, Klf4, and c-Myc (Takahashiand Yamanaka, 2006).

Subsequent studies have shown that human iPS cells can be obtained usingsimilar transduction methods and the transcription factor quartet, OCT4,SOX2, LIN28 and NANOG. The production of iPS cells can be achieved bythe introduction of nucleic acid sequences encoding stem cell-associatedgenes into an adult, somatic cell, historically using viral vectors.

iPS cells can be generated or derived from terminally differentiatedsomatic cells, as well as from adult stem cells, or somatic stem cells.That is, a non-pluripotent progenitor cell can be rendered pluripotentor multipotent by reprogramming. In such instances, it may not benecessary to include as many reprogramming factors as required toreprogram a terminally differentiated cell. Further, reprogramming canbe induced by the non-viral introduction of reprogramming factors, e.g.,by introducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 2010 Nov. 5; 7(5):618-30). Reprogrammingcan be achieved by introducing combinations of nucleic acids encodingstem cell-associated genes including, for example Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. In oneembodiment, reprogramming using the methods and compositions describedherein can comprise introducing one or more of Oct-3/4, a member of theSox family, a member of the Klf family, and a member of the Myc familyto a somatic cell. In one embodiment, the methods and compositionsdescribed herein further comprise introducing one or more of each ofOct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. In someembodiments, the reprogramming can occur in the absence of forcedexpression of c-Myc. Methods and factors for reprogramming are reviewedby Theunissen and Jaenisch (2014) Cell-Stem Cell 14:720-734.

As noted above, the exact method used for reprogramming is notnecessarily critical to the methods and compositions described herein.However, where cells differentiated from the reprogrammed cells are tobe used in, e.g., human therapy, in one embodiment the reprogramming isnot effected by a method that alters the genome. Thus, in suchembodiments, reprogramming is achieved, e.g., without the use of viralor plasmid vectors. In addition to the protein-based and the RNA-basedmethods (see e.g., Warren et al., supra), recent evidence indicatessomatic cells may be re-programmed by e.g., exposure of the cells tounphysiological stress, e.g., in culture (see e.g., WO2013/163296, whichis incorporated herein by reference in its entirety). Methods such asthese that do not permanently modify the genome may be preferred forcells to be used for therapeutic purposes, as they are less likely toprovoke genomic damage likely to promote, e.g., cancer.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various small molecules, as shown by Shi, Y., et al (2008)Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3:132-135. Anagent or combination of agents that enhance the efficiency or rate ofinduced pluripotent stem cell production can be helpful in anyreprogramming situation, but can be particularly advantageous, forexample, in the production of patient-specific or disease-specificiPSCs. In addition to the factors disclosed herein, some non-limitingexamples of agents that enhance reprogramming efficiency include solubleWnt, Wnt conditioned media, BIX-01294 (a G9a histone methyltransferase),PD0325901 (a MEK inhibitor), DNA methyltransferase inhibitors, histonedeacetylase (HDAC) inhibitors, valproic acid, 5′-azacytidine,dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, andtrichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VPA)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE(2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Otherreprogramming enhancing agents include, for example, dominant negativeforms of the HDACs (e.g., catalytically inactive forms), siRNAinhibitors of the HDACs, and antibodies that specifically bind to theHDACs. Such inhibitors are available, e.g., from BIOMOL International,Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, AtonPharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, andSigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto,Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, a cellthat expresses Oct4 or Nanog is identified as pluripotent. Methods fordetecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses. In someembodiments, detection does not involve only RT-PCR, but also includesdetection of protein markers. Intracellular markers may be bestidentified via RT-PCR, while cell surface markers are readilyidentified, e.g., by immunocytochemistry. It can also be advantageous inappropriate circumstances to use a reporter construct, e.g., driven bythe regulatory elements for a stem cell marker to identify areprogrammed cell.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate to cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells are introduced to nude mice and histologyand/or immunohistochemistry is performed on a tumor arising from thecells. The growth of a tumor comprising cells from all three germlayers, for example, further indicates that the cells are pluripotentstem cells.

Further, cells undergoing reprogramming pass through definedintermediate stages that can be tracked and isolated based oncombinations of surface markers and reporter alleles (see e.g., Polo etal. (2012) Cell 151:1617-1632; Stadtfeld et al. (2008) Cell Stem Cell2:23-240). Briefly, cells initially downregulate THY1 and thenupregulate SSEA1, followed by EPCAM and eventually Oct4 (or PECAM)activation. Only cells that follow this transition in a timely mannerwill give rise to iPSCs. Thus, one of skill in the art can assesschanges in these surface markers and reporter alleles to preciselydetect or confirm the process of reprogramming.

Somatic Cells for Reprogramming to iPSCs:

Somatic cells, as that term is used herein, refers to any cells formingthe body of an organism, excluding germline cells. Every cell type inthe mammalian body—apart from the sperm and ova, the cells from whichthey are made (gametocytes) and undifferentiated stem cells—is adifferentiated somatic cell. For example, internal organs, skin, bones,blood, and connective tissue are all made up of differentiated somaticcells. It is contemplated herein that somatic cells from any of thesetissues can be reprogrammed while taking advantage of the methods andcompositions described herein.

Some non-limiting examples of differentiated somatic cells include, butare not limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, immune cells, hepatic, splenic, lung, circulating bloodcells, gastrointestinal, renal, bone marrow, and pancreatic cells. Insome embodiments, a somatic cell can be a primary cell isolated from anysomatic tissue including, but not limited to brain, liver, lung, gut,stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ,bone, etc. Further, the somatic cell can be from any mammalian species,with non-limiting examples including a murine, bovine, simian, porcine,equine, ovine, or human cell. In some embodiments, the somatic cell is ahuman somatic cell.

When reprogrammed cells are used in the therapeutic treatment ofdisease, it is desirable, but not required, to use somatic cellsisolated from the patient being treated. In some embodiments, a methodfor selecting the reprogrammed cells from a heterogeneous populationcomprising reprogrammed cells and somatic cells they were derived orgenerated from can be performed by any known means. For example, a drugresistance gene or the like, such as a selectable marker gene can beused to isolate the reprogrammed cells using the selectable marker as anindex. It is emphasized that such a selectable marker is notrequired—that is, in some embodiments reprogrammed pluripotent stemcells can be identified on the basis of morphology (see e.g.,US2010/0184051).

Transdifferentiation

Transdifferentiation, also known as lineage reprogramming or directconversion, is a process where cells convert from one differentiatedcell type to another without undergoing an intermediate pluripotentstate or progenitor cell type. Transdifferentiation has been proposed asan approach for disease modeling, drug discovery, gene therapy andregenerative medicine.

Provided herein are methods for inducing transdifferentiation thatinclude inhibiting or depleting CAF-1. Effects of CAF-1 inhibitiontreatment on transdifferentiation can be assessed during“fibroblast-to-neuron” or “B-cell to macrophage” transdifferentiationusing e.g., defined transcription factors as detailed in the Examplesherein. However, the effects of CAF-1 inhibition are expected to bebroadly applicable to any cell transdifferentiation, particularly sinceCAF-1-mediated in chromatin assembly and DNA packaging necessarily occurin all cell types.

Transdifferentiation can be performed with the methods and compositionsdescribed herein, using any means known in the art. In one approach,termed the “lineage instructive approach” transcription factors fromprogenitor cells of the target cell type are transfected into a somaticcell to induce transdifferentiation. One of skill in the art candetermine which transcription factors to use by starting with a largepool of factors and narrowing down to a specific target cell typetranscription factor, or vice versa.

Another approach to transdifferentiation is the “initial epigeneticactivation phase approach,” where somatic cells are first transfectedwith pluripotent reprogramming factors temporarily (Oct4, Sox2, Nanog,etc.) before being transfected with the desired inhibitory or activatingfactors.

Transdifferentiation can also be induced using pharmacological agents,particularly demethylating agents such as 5-azacytidine, or5-aza-2-deoxycytidine, zebularine, procaine, epigallocatechin-3-gallate,RG108, 1-β-D-arabinofuranosyl-5-azacytosine, dihydro-5-azacytidine orL-ethionine. 5-azacytidine has been shown to promote phenotypictransdifferentiation of cardiac cells to skeletal myoblasts. In someembodiments, growth factors can be included during transdifferentiation,Exemplary growth factors include granulocyte-macrophage stimulatingfactor (GM-CSF), stem cell factor (SCF), G-CSF, M-CSF, thrombopoietin,IL-2, IL-4, fibroblast growth factor (FGF), epidermal growth factor(EGF) and/or vascular endothelial growth factor. In some embodiments,more than one growth factor will be included.

Further examples of methods for transdifferentiation can be found ine.g., Vierbuchen and Wernig (2012) Molecular Cell 47:827-838.

Successful transdifferentiation can be determined using the presence orthe absence of the expression of a marker that is specific to a targetcell as an indicator. The expression of such marker can be detected bybiochemical or immunochemical techniques known by persons skilled in theart. For example, immunochemical techniques such as enzyme-linkedimmunosorbent assay (ELISA), immunofluorescent assay (IFA),immunoelectrophoresis, immunochromatography assay, andimmunohistochemical staining method can be used. In these methods,marker-specific polyclonal or monoclonal antibodies or fragmentsthereof, which selectively bind to antigens of various target cells, areused. Antibodies against various specific markers are marketed and canbe properly obtained by persons skilled in the art. The expression of amarker that is specific to a target cell can also be detected by amolecular biological method such as RT-PCR, transcription mediatedamplification (TMA), reverse transcriptase-ligase chain reaction(RT-LCR), or hybridization analysis. Alternatively, the expression ofsuch marker can also be specifically detected by a tissue stainingtechnique such as alizarin red staining, alcian blue staining, Oil-Red-Ostaining, Von Kossa staining, or indocyanine green staining usingmetabolic products of differentiated cells, cellular drug metabolism, orproperties such as dyeing properties (e.g., a dye exclusion assay).

CAF-1 Complex

DNA is packaged into chromatin, in part to control gene expression. Inorder for DNA replication to occur, chromatin is disassembled andnucleosomes are transiently removed from the DNA strand. Nucleosomesquickly reassemble behind the DNA replication fork to re-package theDNA. New nucleosomes are generated at the replication fork in a reactioncatalyzed by chromatin assembly factor 1 (CAF-1; NCBI Gene ID: 41836).CAF-1 consists of three polypeptides with molecular weights of 150(i.e., Chaf1a; NCBI Gene ID: 10036), 60 (i.e., Chaf1b; NCBI Gene ID:8208), and 48 kDa that bind histones H3 and H4. Inhibition of theexpression or activity of any or all of these subunits or an inhibitorthat targets the complex (or subunits thereof) can be employed in themethods and compositions disclosed herein.

CAF-1 is also thought to play a role in depositing the histone tetrameronto replicating DNA. In addition, CAF-1 has been associated with therestoration of chromatin structure after DNA repair, and with themaintenance of heterochromatin.

Sumo2

Small ubiquitin-like modifier (Sumo) proteins are a group of smallproteins that bind lysine residues of target proteins and thereby modifytarget protein activity, stability, and sub-cellular localization. Thereare several different Sumo isoforms, including Sumo1, Sumo2 (NCBI GeneID: 6613), and Sumo 3. Sumo2 and Sumo3 proteins share a high degree ofsimilarity (95% sequence identity), but are relatively distinct fromSumo1 (only 50% sequence identity). Like ubiquitin, the Sumo protein issynthesized as a larger precursor protein that is processed bysentrin-specific proteases to expose the two C-terminal glycine residuesthat provide for conjugation. Sumo conjugation (or “sumoylation”) is ahighly volatile process, with various enzymes involved in theconjugation, e.g., E1, E2 and Ubc9, and de-conjugating (or“de-sumoylation”) e.g., SENPs, processes. A number of known Sumoconjugation targets transcription factors and other nuclear proteinsinvolved in gene expression. A major change in levels of Sumo conjugatedproteins may have a major impact on the fate of cells.

Nutd21

The Nudix (nucleoside diphosphate linked moiety X)-type motif 21 gene(NCBI Gene ID: 11051) encodes the protein “cleavage and polyadenylationspecificity factor subunit 5.” The gene product is one subunit of acleavage factor required for 3′ RNA cleavage and polyadenylationprocessing. Interaction of the protein with RNA is one of the earlieststeps in the assembly of the 3′ end processing complex and facilitatesthe recruitment of other processing factors. This gene encodes the 25kDsubunit of the protein complex, which is composed of four polypeptides.

Inhibitors of CAF-1, Sumo2 and Nutd21

Essentially any inhibitor of CAF-1, Sumo2, and/or Nutd21 can be usedwith the methods and treatments described herein. An inhibitor can be anRNA interference agent (e.g., shRNA, siRNA), an antibody or antigenbinding agent, a small molecule, etc. One of skill in the art canreadily identify inhibitors of CAF-1, Sumo2, and/or Nutd21 usingcellular screening assays that are routine in the art.

As used herein, the term “inhibitor of the CAF-1 complex, Sumo2 orNutd1” refers to a molecule or agent that significantly blocks,inhibits, reduces, or interferes with the CAF-1 complex, Sumo2, or Nutd1biological activity in vitro, in situ, and/or in vivo, includingactivity of downstream pathways mediated by CAF-1, Sumo2, or Nutd1signaling, such as, for example, RNA or protein upregulation, and/orelicitation of a cellular response to CAF-1, Sumo2, or Nutd1. Exemplaryinhibitors contemplated for use in the various aspects and embodimentsdescribed herein include, but are not limited to, antibodies orantigen-binding fragments thereof that specifically bind to one or moremembers of the CAF-1 complex, Sumo2, Nutd1 or their isoforms; anti-sensemolecules directed to a nucleic acid encoding one of the targets; shortinterfering RNA (“siRNA”) molecules directed to a nucleic acid encodinga member of the CAF-1 complex, Sumo2, or Nutd21; an inhibitory compound;RNA or DNA aptamers that bind one or more members of the CAF-1 complex,Sumo2, Nutd1 or their isoforms, and inhibit/reduce/block activity orsignaling; inhibitory soluble CAF-1, Sumo2, or Nutd21 proteins or fusionpolypeptides thereof (e.g., dominant negative mutants).

As used herein, an inhibitor or antagonist has the ability to reduce theactivity and/or expression of the CAF-1 complex, Sumo2, or Nutd21 in acell by at least 5%, at least 10%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, at least 99%, or more, relative to theactivity or expression level in the absence of the inhibitor orantagonist. At a minimum, an inhibitor will interfere with theinhibitory effect of these factors in a reprogramming assay as describedin the Examples—that is, an inhibitor will increase the speed orefficiency of reprogramming in an assay as described herein. In someembodiments, the activity or expression of the CAF-1 complex can beassessed using a commercial ELISA kit (e.g., from LIFESPAN BIOSCIENCES)or as described in Verreault et al. (1996) 87(1):95-104. In someembodiments, the activity or expression of Sumo2 can be assessed using acommercial Sumoylation assay kit, for example, Sumo2-FP (UBIQ™), orSUMOylation assay kit (ABCAM™). In some embodiments, the activity ofexpression of Nutd21 can be assessed using Real-Time PCR primers frome.g., BIORAD, TAQMAN, etc.

In some embodiments, particularly with regard to inhibition of the CAF-1complex, it is not desirable to inhibit the target completely. One ofskill in the art can readily determine or titrate a dose of theinhibitor to achieve a desired result to be used in combination with acellular reprogramming protocol or for administration to a subject.

RNA Interference Agents:

The use of RNA interference agents is well within the abilities of oneof skill in the art and is not described in detail herein. RNAinterference (RNAi) uses e.g., small interfering RNA (siRNA) duplexesthat target the messenger RNA encoding the target polypeptide forselective degradation via the RNA-induced silencing complex (RISC).siRNA-dependent post-transcriptional silencing of gene expressioninvolves cleaving the target messenger RNA molecule at a site guided bye.g., the siRNA. As used herein, “inhibition of target gene expression”includes any decrease in expression or protein activity or level of thetarget gene or protein encoded by the target gene as compared to asituation wherein no RNA interference has been induced. The decreasewill be of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or99% or more as compared to the expression of a target gene or theactivity or level of the protein encoded by a target gene which has notbeen targeted by an RNA interfering agent.

The terms “RNA interference agent” and “RNA interference” can comprise ashort interfering RNA (siRNA), miRNA, shRNA or other double-stranded RNAmolecule that targets a gene of interest. Such agents can be chemicallysynthesized, produced by in vitro transcription, or produced within ahost cell. In one embodiment, siRNA is a double stranded RNA (dsRNA)molecule of about 15 to about 40 nucleotides in length, preferably about15 to about 28 nucleotides, more preferably about 19 to about 25nucleotides in length, and more preferably about 19, 20, 21, 22, or 23nucleotides in length, and may contain a 3′ and/or 5′ overhang on eachstrand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. Thelength of the overhang is independent between the two strands, i.e., thelength of the overhang on one strand is not dependent on the length ofthe overhang on the second strand. Preferably the siRNA is capable ofpromoting RNA interference through degradation or specificpost-transcriptional gene silencing (PTGS) of the target messenger RNA(mRNA).

siRNAs also include small hairpin (also called stem loop) RNAs (shRNAs).In one embodiment, shRNAs are composed of a short (e.g., about 19 toabout 25 nucleotide) antisense strand, followed by a nucleotide loop ofabout 5 to about 9 nucleotides, and the analogous sense strand.Alternatively, the sense strand may precede the nucleotide loopstructure and the antisense strand may follow. Sequences encoding theseshRNAs can be contained in plasmids, retroviruses, and lentiviruses andexpressed from, for example, the pol III U6 promoter, or anotherpromoter. The target gene or sequence of the RNA interfering agent canbe a cellular gene or genomic sequence, e.g. a Chaf1a, Chaf1b, Sumo2, orNutd21 sequence. An siRNA can be substantially homologous to the targetgene or genomic sequence, or a fragment thereof. As used in thiscontext, the term “homologous” is defined as being substantiallyidentical, sufficiently complementary, or similar to the target mRNA, ora fragment thereof, to effect RNA interference of the target. Inaddition to native RNA molecules, RNA suitable for inhibiting orinterfering with the expression of a target sequence includes RNAderivatives and analogs that permit RNA-mediated gene silencing.Preferably, one strand of the siRNA is identical to its target. ThesiRNA preferably targets only one sequence. Each of the RNA interferingagents, such as siRNAs, can be screened for potential off-target effectsby, for example, expression profiling. Such methods are known to oneskilled in the art. In addition to expression profiling, one can alsoscreen the potential target sequences for similar sequences in thesequence databases to identify potential sequences which may haveoff-target effects. Therefore, one may initially screen the proposedsiRNAs to avoid potential off-target silencing using sequence identityanalysis by any known sequence comparison methods, such as BLAST. siRNAsequences can also be chosen to maximize the uptake of the antisense(guide) strand of the siRNA into RISC and thereby maximize the abilityof RISC to target an mRNA for degradation. siRNA molecules need not belimited to those molecules containing only RNA, but, for example,further encompasses chemically modified nucleotides and non-nucleotides,and also include molecules wherein a ribose sugar molecule issubstituted for another sugar molecule or a molecule which performs asimilar function.

siRNA sequences to target the CAF-1 complex (e.g., Chaf1a, Chaf1b etc.),Sumo2, Nutd21, can also be obtained commercially from e.g., INVITROGEN™,THERMO SCIENTIFIC™, and ORIGENE™, among others.

Antibodies and Antigen Binding Agents:

Antibodies for binding and/or inhibition of a member of the CAF-1complex, Sumo2, or Nutd21 suitable for use in practicing the methodsdescribed herein are preferably monoclonal, and can include, but are notlimited to, human, humanized or chimeric antibodies, comprising singlechain antibodies, Fab fragments, F(ab′) fragments, fragments produced bya Fab expression library, and/or binding fragments of any of the above.Antibodies also refer to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain antigen or target binding sites or “antigen-binding fragments.”The immunoglobulin molecules described herein can be of any type (e.g.,IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2) or subclass of immunoglobulin molecule, as is understoodby one of skill in the art.

It is noted that antibodies are widely perceived to be ineffective fortargeting intracellular proteins due to relative difficulty in crossingcellular membranes. However, recent evidence indicates that unmodifiedantibodies can cross the plasma or cell membrane to bind and inhibitintracellular targets (see e.g., US 2014/0286937; Guo et al. (2011)Science Translational Medicine 3:99ra85). As such, antibodies orantibody fragments as known in the art and as described herein can beused to inhibit intracellular factors, including CAF-1 complex, Sumo2and Nutd21. Examples of antibody fragments encompassed by the termsantibody fragment or antigen-binding fragment include: (i) the Fabfragment, having V_(L), C_(L), V_(H) and C_(H)1 domains; (ii) the Fab′fragment, which is a Fab fragment having one or more cysteine residuesat the C-terminus of the C_(H)1 domain; (iii) the Fd fragment havingV_(H) and C_(H)1 domains; (iv) the Fd′ fragment having V_(H) and C_(H)1domains and one or more cysteine residues at the C-terminus of theC_(H)1 domain; (v) the Fv fragment having the V_(L) and V_(H) domains ofa single arm of an antibody; (vi) a dAb fragment, which consists of aV_(H) domain or a V_(L) domain; (vii) isolated CDR regions; (viii)F(ab′)₂ fragments, a bivalent fragment including two Fab′ fragmentslinked by a disulphide bridge at the hinge region; (ix) single chainantibody molecules (e.g. single chain Fv; scFv), (x) “diabodies” withtwo antigen binding sites, comprising a heavy chain variable domain(V_(H)) connected to a light chain variable domain (V_(L)) in the samepolypeptide chain, (xi) “linear antibodies” comprising a pair of tandemFd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which, together withcomplementary light chain polypeptides, form a pair of antigen bindingregion; and modified versions of any of the foregoing (e.g., modified bythe covalent attachment of polyalkylene glycol (e.g., polyethyleneglycol, polypropylene glycol, polybutylene glycol) or other suitablepolymer). Although CAF-1, Sumo2, and Nutd21 are intracellular proteins,it is now accepted that antibody agents can be used to targetintracellularly (see e.g., U.S. Pat. No. 8,715,674).

Small Molecule Inhibitors:

In some embodiments of the compositions, methods, and uses describedherein, an inhibitor of the CAF-1 complex, Sumo2, or Nutd21 is a smallmolecule inhibitor, including, but not limited to, small peptides orpeptide-like molecules, soluble peptides, and synthetic non-peptidylorganic or inorganic compounds. A small molecule inhibitor or antagonistcan have a molecular weight of any of about 100 to about 20,000 daltons(Da), about 500 to about 15,000 Da, about 1000 to about 10,000 Da. Insome embodiments, an inhibitor comprises a small molecule that binds toa member of the CAF-1 complex, Sumo2, or Nutd21 and inhibits itsbiological activity.

Combination Treatment:

In some embodiments, inhibitors of the CAF-1 complex, Sumo2, and Nutd21can be used in combination. For example, inhibitors targeting at leasttwo of the CAF-1 complex, Sumo2, and/or Nutd21 can be used incombination. In other embodiments, inhibitors targeting all three of theCAF-1 complex, Sumo2, and Nutd21 can be used in combination.

Promoting Cancer Cell Differentiation

Germline deletion of CAF-1 in mice results in early embryonic lethality,whereas CAF-1 loss in embryonic stem cells causes cell cycle arrest andapoptosis. In the working examples, CAF-1 is shown to act as a generalstabilizer of cell identity during normal development anddifferentiation, in part by maintaining epigenetic patterns in somaticcells. CAF-1 depletion in several pre-leukemic tumor cell lines, whichwere generated by viral expression of HoxA9, or HoxB8 in myeloidprogenitor cells, triggered differentiation and subsequently growtharrest.

Accordingly, inhibitors of CAF-1 can be used to promote differentiationof a cancer cell in vivo. In other embodiments, a CAF-1 inhibitor isadministered to a subject for the treatment of cancer. It is expectedthat a decrease of CAF-1 expression and/or activity will be effectiveagainst any cancer type that involves cancer stem cells. Cancers ingeneral often behave like earlier developmental forms of the tissuesfrom which they originate, such that promotion of differentiation willlimit the uncontrolled proliferation that is characteristic of cancer.As such, inhibition of CAF-1 is expected to do so with broadapplicability.

Some non-limiting examples of cancer that can be treated with a CAF-1inhibitor include, but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. Other exemplary cancers include, butare not limited to, basal cell carcinoma, biliary tract cancer; bladdercancer; bone cancer; brain and CNS cancer; breast cancer; cancer of theperitoneum; cervical cancer; choriocarcinoma; colon and rectum cancer;connective tissue cancer; cancer of the digestive system; endometrialcancer; esophageal cancer; eye cancer; cancer of the head and neck;gastric cancer (including gastrointestinal cancer); glioblastoma;hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renalcancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g.,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, and squamous carcinoma of the lung); lymphoma includingHodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma;oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovariancancer; pancreatic cancer; prostate cancer; retinoblastoma;rhabdomyosarcoma; rectal cancer; cancer of the respiratory system;salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer;stomach cancer; testicular cancer; thyroid cancer; uterine orendometrial cancer; cancer of the urinary system; vulval cancer; as wellas other carcinomas and sarcomas; as well as B-cell lymphoma (includinglow grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic(SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuseNHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; highgrade small non-cleaved cell NHL; bulky disease NHL; mantle celllymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia);chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL);Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome.

In some embodiments, the carcinoma or sarcoma includes, but is notlimited to, carcinomas and sarcomas found in the anus, bladder, bileduct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus,eye, gallbladder, head and neck, liver, kidney, larynx, lung,mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin,small intestine, stomach, spinal marrow, tailbone, testicles, thyroidand uterus. The types of carcinomas include but are not limited topapilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma,adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma,rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma,lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, largecell undifferentiated carcinomas, basal cell carcinoma and sinonasalundifferentiated carcinoma. The types of sarcomas include but are notlimited to, for example, soft tissue sarcoma such as alveolar soft partsarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplasticsmall round cell tumor, extraskeletal chondrosarcoma, extraskeletalosteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma,Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma,rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma(primitive neuroectodermal tumor), malignant hemangioendothelioma,malignant schwannoma, osteosarcoma, and chondrosarcoma.

In one embodiment of the methods, the subject having the tumor, canceror malignant condition is undergoing, or has undergone, treatment with aconventional cancer therapy. In some embodiments, the cancer therapy ischemotherapy, radiation therapy, immunotherapy or a combination thereof.

Inhibitors of CAF-1 can be used alone or in combination with othertherapies, including chemotherapy, radiation, cancer immunotherapy, orcombinations thereof. Such therapies can either directly target a tumor(e.g., by inhibition of a tumor cell protein or killing of highlymitotic cells) or provoke or accentuate an anti-tumor immune response.

Exemplary anti-cancer agents that can be used in combination with aCAF-1 inhibitor include alkylating agents such as thiotepa and CYTOXAN™;cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas,such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaI1); dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™,doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK. polysaccharidecomplex (JHS Natural Products™, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL™paclitaxel (Bristol-Myers Squibb™ Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners™, Schaumberg, Ill.), andTAXOTERE™ doxetaxel (Rhone-Poulenc Rorer™, Antony, France);chloranbucil; GEMZAR™, gemcitabine; 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin, oxaliplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; NAVELBINE™, vinorelbine; novantrone;teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate;irinotecan (Camptosar, CPT-11) (including the treatment regimen ofirinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid;capecitabine; combretastatin; leucovorin (LV); oxaliplatin, includingthe oxaliplatin treatment regimen (FOLFOX™); lapatinib (Tykerb™);inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva™))and VEGF-A that reduce cell proliferation and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. In addition,the methods of treatment can further include the use of radiation.

Dosage and Administration

In some aspects, the methods described herein provide a method forinducing cellular differentiation in vivo or a method for treatingcancer in a subject. In one embodiment of this aspect and all otheraspects described herein, the cancer is leukemia. In one embodiment, thesubject can be a mammal. In another embodiment, the mammal can be ahuman, although the approach is effective with respect to all mammals.The methods comprise administering to the subject an effective amount ofa pharmaceutical composition comprising an inhibitor that binds a memberof the CAF-1 complex (e.g., Chaf1a, Chaf1b), or a combination thereof ina pharmaceutically acceptable carrier.

The dosage range for the agent depends upon the potency, and includesamounts large enough to produce the desired effect, e.g., cellulardifferentiation or treatment of cancer. The dosage should not be solarge as to cause unacceptable adverse side effects. Generally, thedosage will vary with the type of inhibitor (e.g., an antibody orfragment, small molecule, siRNA, etc.), and with the age, condition, andsex of the patient. The dosage can be determined by one of skill in theart and can also be adjusted by the individual physician in the event ofany complication.

Typically, the dosage ranges from 0.001 mg/kg body weight to 5 g/kg bodyweight. In some embodiments, the dosage range is from 0.001 mg/kg bodyweight to 1 g/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kgbody weight, from 0.001 mg/kg body weight to 0.1 g/kg body weight, from0.001 mg/kg body weight to 50 mg/kg body weight, from 0.001 mg/kg bodyweight to 25 mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kgbody weight, from 0.001 mg/kg body weight to 5 mg/kg body weight, from0.001 mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg bodyweight to 0.1 mg/kg body weight, from 0.001 mg/kg body weight to 0.005mg/kg body weight. Alternatively, in some embodiments the dosage rangeis from 0.1 g/kg body weight to 5 g/kg body weight, from 0.5 g/kg bodyweight to 5 g/kg body weight, from 1 g/kg body weight to 5 g/kg bodyweight, from 1.5 g/kg body weight to 5 g/kg body weight, from 2 g/kgbody weight to 5 g/kg body weight, from 2.5 g/kg body weight to 5 g/kgbody weight, from 3 g/kg body weight to 5 g/kg body weight, from 3.5g/kg body weight to 5 g/kg body weight, from 4 g/kg body weight to 5g/kg body weight, from 4.5 g/kg body weight to 5 g/kg body weight, from4.8 g/kg body weight to 5 g/kg body weight. In one embodiment, the doserange is from 5 μg/kg body weight to 30 μg/kg body weight.Alternatively, the dose range will be titrated to maintain serum levelsbetween 5 μg/mL and 30 μg/mL.

Administration of the doses recited above can be repeated for a limitedperiod of time. In some embodiments, the doses are given once a day, ormultiple times a day, for example but not limited to three times a day.In a preferred embodiment, the doses recited above are administereddaily for several weeks or months. The duration of treatment dependsupon the subject's clinical progress and responsiveness to therapy.Continuous, relatively low maintenance doses are contemplated after aninitial higher therapeutic dose.

A therapeutically effective amount is an amount of an agent that issufficient to produce a statistically significant, measurable change inimmune response (see “Efficacy Measurement” below). Such effectiveamounts can be gauged in clinical trials as well as animal studies for agiven agent.

Agents useful in the methods and compositions described herein can beadministered topically, intravenously (by bolus or continuous infusion),orally, by inhalation, intraperitoneally, intramuscularly,subcutaneously, intracavity, and can be delivered by peristaltic means,if desired, or by other means known by those skilled in the art. Theagent can be administered systemically, if so desired.

Therapeutic compositions containing at least one agent can beconventionally administered in a unit dose. The term “unit dose” whenused in reference to a therapeutic composition refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredphysiologically acceptable diluent, i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. An agent can be targeted by meansof a targeting moiety, such as e.g., an antibody or targeted liposometechnology. In some embodiments, an agent can be targeted to a tissue byusing bispecific antibodies, for example produced by chemical linkage ofan anti-ligand antibody (Ab) and an Ab directed toward a specifictarget. To avoid the limitations of chemical conjugates, molecularconjugates of antibodies can be used for production of recombinantbispecific single-chain Abs directing ligands and/or chimeric inhibitorsat cell surface molecules. The addition of an antibody to an agentpermits the agent to accumulate additively at the desired target site(e.g., a tumor). Antibody-based or non-antibody-based targeting moietiescan be employed to deliver a ligand or the inhibitor to a target site.Preferably, a natural binding agent for an unregulated or diseaseassociated antigen is used for this purpose.

Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner and are particular to eachindividual. However, suitable dosage ranges for systemic application aredisclosed herein and depend on the route of administration. Suitableregimes for administration are also variable, but are typified by aninitial administration followed by repeated doses at one or moreintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

Pharmaceutical Compositions

The present disclosure includes, but is not limited to, therapeuticcompositions useful for practicing the therapeutic methods describedherein. Therapeutic compositions contain a physiologically tolerablecarrier together with an active agent as described herein, dissolved ordispersed therein as an active ingredient. In a preferred embodiment,the therapeutic composition is not immunogenic when administered to amammal or human patient for therapeutic purposes. As used herein, theterms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a mammal without theproduction of undesirable physiological effects such as nausea,dizziness, gastric upset and the like. A pharmaceutically acceptablecarrier will not promote the raising of an immune response to an agentwith which it is admixed, unless so desired. The preparation of apharmacological composition that contains active ingredients dissolvedor dispersed therein is well understood in the art and need not belimited based on formulation. Typically such compositions are preparedas injectable either as liquid solutions or suspensions, however, solidforms suitable for solution, or suspensions, in liquid prior to use canalso be prepared. The preparation can also be emulsified or presented asa liposome composition. The active ingredient can be mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient and in amounts suitable for use in the therapeuticmethods described herein. Suitable excipients include, for example,water, saline, dextrose, glycerol, ethanol or the like and combinationsthereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents and the like which enhance the effectiveness of theactive ingredient. The therapeutic composition of the present inventioncan include pharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active agent used in the methodsdescribed herein that will be effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques.

Efficacy Measurement

The efficacy of a given treatment for a cancer (including, but notlimited to, leukemia) can be determined by the skilled clinician.However, a treatment is considered “effective treatment,” as the term isused herein, if any one or all of the signs or symptoms of the canceris/are altered in a beneficial manner, or other clinically acceptedsymptoms or markers of disease are improved, or ameliorated, e.g., by atleast 10% following treatment with an agent that comprises an inhibitorthat binds a member of the CAF-1 complex, Sumo2, or Nutd21. Efficacy canalso be measured by failure of an individual to worsen as assessed bystabilization of the disease, or the need for medical interventions(i.e., progression of the disease is halted or at least slowed). Methodsof measuring these indicators are known to those of skill in the artand/or described herein. Treatment includes any treatment of a diseasein an individual or an animal (some non-limiting examples include ahuman, or a mammal) and includes: (1) inhibiting the disease, e.g.,arresting, or slowing progression of the cancer; or (2) relieving thedisease, e.g., causing regression of symptoms; and (3) preventing orreducing the likelihood of the development of the disease, or preventingsecondary diseases/disorders associated with the cancer (e.g., cancermetastasis).

An effective amount for the treatment of a disease means that amountwhich, when administered to a mammal in need thereof, is sufficient toresult in effective treatment as that term is defined herein, for thatdisease. Efficacy of an agent can be determined by assessing physicalindicators of the disease, such as e.g., pain, tumor size, tumor growthrate, blood cell count, etc.

Kits

Also provided herein are kits for inducing cellular reprogramming. At aminimum, the kit(s) described herein comprise at least one inhibitor ofthe CAF-1 complex, Sumo2, or Nutd21. In one embodiment, the inhibitorcomprises an inhibitor of Chaf1a and/or Chaf1b. The inhibitor can be anRNA interference agent, an antibody or antigen binding agent, a smallmolecule, an aptamer, an antisense molecule, a dominant negative etc.The kit can comprise a single vial of the inhibitor or can compriseindividual aliquots (e.g., a unit dose).

In some embodiments, the kits comprise at least two inhibitors, at least3, at least 4, at least 5 or more. In some embodiments, the plurality ofinhibitors are packaged in separate vials. In other embodiments, theplurality of inhibitors are formulated together as a cocktail.

The kit may also comprise components for inducing cellularreprogramming, such as at least one virus, plasmid or vector comprisinga nucleic acid sequence encoding a reprogramming factor (e.g., Oct4,Sox2, Klf4, c-Myc, Lin-28, Nanog, etc.). In some embodiments, the kitcomprises one or more agents known to enhance efficiency ofreprogramming. In some embodiments, the kit further comprises cellgrowth media and/or reprogramming media.

In some embodiments, the kit comprises primers for detecting cellsurface markers on reprogrammed cells.

The kit will typically be provided with its various elements included inone package, e.g., a fiber-based, e.g., a cardboard, or polymeric, e.g.,a Styrofoam box. The enclosure can be configured so as to maintain atemperature differential between the interior and the exterior, e.g., itcan provide insulating properties to keep the reagents at a preselectedtemperature for a preselected time. Instructions for use of thecomponents of the kit is packaged therein.

The present invention may be as defined in any one of the followingnumbered paragraphs or in any combination of the following numberedparagraphs.

1. A method for performing cellular reprogramming, the methodcomprising:

(a) contacting a somatic cell with an inhibitor of the CAF-1 complex,Nudt21, or Sumo2, and (b) subjecting the somatic cell to a reprogrammingprotocol, thereby reprogramming the somatic cell to an inducedpluripotent stem cell (iPSC).

2. The method of paragraph 1, wherein the speed and/or efficiency ofcellular reprogramming to iPSCs is increased in the presence of theinhibitor as compared to the speed and/or efficiency of cellularreprogramming performed in the absence of the inhibitor.

3. The method of paragraph 1 or 2, wherein the measure of efficiency ofcellular reprogramming comprises an increase in the total number ofreprogrammed cells relative to reprogramming in the absence of a theinhibitor.

4. The method of paragraph 1, 2 or 3, wherein the measure of speed ofcellular reprogramming comprises the appearance of reprogrammed cells atan earlier time point than occurs when reprogramming in the absence ofthe inhibitor.

5. The method of any one of paragraphs 1-4, wherein the inhibitorcomprises an RNA interference molecule or an antibody.

6. The method of any one of paragraphs 1-5, wherein the RNA interferencemolecule comprises an siRNA or an shRNA.

7. The method of any one of paragraphs 1-6, wherein step (a) isperformed before or during step (b).

8. The method of any one of paragraphs 1-7, wherein the reprogramming ofstep (b) comprises induction of Oct-4/Klf4/Sox-2/c-Myc (OKSM)expression.

9. The method of any one of paragraphs 1-8, wherein the reprogrammingstep does not comprise forced expression of c-Myc.

10. The method of any one of paragraphs 1-9, wherein the somatic cellcomprises a fibroblast.

11. The method of any one of paragraphs 1-10, wherein the inhibitor ofthe CAF-1 complex inhibits the Chaf1a and/or Chaf1b subunit of thecomplex.

12. A method of inducing differentiation of a cancer cell or cancer stemcell in vivo, the method comprising: administering an inhibitor of theCAF-1 complex to a subject having, or suspected of having cancer,thereby inducing differentiation of the cancer cell or cancer stem cellin vivo.

13. The method of paragraph 12, wherein the cancer comprises leukemia.

14. The method of paragraph 12 or 13, wherein the inhibitor comprises anRNA interference molecule or an antibody.

15. The method of paragraph 12, 13, or 14, wherein the RNA interferencemolecule comprises an siRNA or an shRNA.

16. The method of any one of paragraphs 12-15, wherein the inhibitorinhibits the Chaf1a and/or Chaf1b subunit of the CAF-1 complex.

17. A composition comprising: one or more inhibitors of the CAF-1complex, Nudt21, or Sumo2 and a pharmaceutically acceptable carrier.

18. The composition of paragraph 17, comprising inhibitors of any two orall of the CAF-1 complex, Nudt21 and Sumo2.

19. A composition for use in cellular reprogramming, the compositioncomprising an inhibitor of the CAF-1 complex, Nudt21, or Sumo2.

20. The composition for use of paragraph 19, further comprising apharmaceutically acceptable carrier.

21. The composition for use of paragraph 19 or 20, wherein the inhibitorincreases the total number of reprogrammed cells relative toreprogramming in the absence of a the inhibitor.

22. The composition for use of paragraph 19, 20 or 21, wherein theinhibitor promotes the appearance of reprogrammed cells at an earliertime point than occurs when cells are reprogrammed in the absence of theinhibitor.

23. The composition for use of any one of paragraphs 19-22, wherein theinhibitor comprises an RNA interference molecule or an antibody.

24. The composition for use of any one of paragraphs 19-23, wherein theRNA interference molecule comprises an siRNA or an shRNA.

25. The composition for use of any one of paragraphs 19-24, whereincellular reprogramming comprises induction of Oct-4/Klf4/Sox-2/c-Myc(OKSM) expression.

26. The composition for use of any one of paragraphs 19-25, whereincellular reprogramming does not comprise forced expression of c-Myc.

27. The composition for use of any one of paragraphs 19-26, whereincellular reprogramming comprises reprogramming of a fibroblast.

28. The composition for use of any one of paragraphs 19-27, wherein theinhibitor of the CAF-1 complex inhibits the Chaf1a and/or Chaf1b subunitof the complex.

29. Use of an inhibitor of the CAF-1 complex, Nudt21, or Sumo2 forcellular reprogramming.

30. The use of paragraph 29, wherein the inhibitor increases the speedand/or efficiency of cellular reprogramming.

31. The use of paragraph 29 or 30, wherein the inhibitor comprises anRNA interference molecule or an antibody.

32. The use of paragraph 29, 30 or 31, wherein the RNA interferencemolecule comprises an siRNA or an shRNA.

33. The use of any one of paragraphs 29-32, wherein cellularreprogramming comprises induction of Oct-4/Klf4/Sox-2/c-Myc (OKSM)expression.

34. The use of any one of paragraphs 29-33, wherein cellularreprogramming does not comprise forced expression of c-Myc.

35. The use of any one of paragraphs 29-34, wherein cellularreprogramming comprises reprogramming of a fibroblast.

36. The use of any one of paragraphs 29-35, wherein the inhibitor of theCAF-1 complex inhibits the Chaf1a and/or Chaf1b subunit of the complex.

37. A composition for use in the treatment of cancer, the compositioncomprising an inhibitor of the CAF-1 complex.

38. The composition for use of paragraph 37, wherein the compositioninduces the differentiation of a cancer cell or cancer stem cell in vivowhen administered to an individual having cancer.

39. The composition for use of paragraph 37 or 38, further comprising apharmaceutically acceptable carrier.

40. The composition for use of paragraph 37, 38, or 39, wherein theinhibitor comprises an RNA interference molecule or an antibody.

41. The composition for use of any one of paragraphs 37-40, wherein theRNA interference molecule comprises an siRNA or an shRNA.

42. The composition for use of any one of paragraphs 37-41, wherein thecancer comprises a leukemia.

43. The composition for use of any one of paragraphs 37-42, wherein theinhibitor inhibits the Chaf1a and/or Chaf1b subunit of the CAF-1complex.

44. Use of an inhibitor of the CAF-1 complex for the treatment ofcancer, the use comprising administering the inhibitor of the CAF-1complex to an individual having cancer.

45. The use of paragraph 44, wherein the administering induces thedifferentiation of a cancer cell or cancer stem cell, thereby treatingthe cancer.

46. The use of paragraph 44 or 45, wherein the inhibitor comprises anRNA interference molecule or an antibody.

47. The use of paragraph 44, 45, or 46, wherein the RNA interferencemolecule comprises an siRNA or an shRNA.

48. The use of any one of paragraphs 44-47, wherein the cancer comprisesa leukemia.

49. The use of any one of paragraphs 44-48, wherein the inhibitorinhibits the Chaf1a and/or Chaf1b subunit of the CAF-1 complex.

50. A method for performing cellular transdifferentiation, the methodcomprising: (a) contacting a somatic cell with an inhibitor of the CAF-1complex, and (b) subjecting the somatic cell to a transdifferentiationprotocol, thereby transdifferentiating the somatic cell to a differentcell type.

51. The method of paragraph 50, wherein the speed and/or efficiency ofcellular transdifferentiation is increased in the presence of theinhibitor as compared to the speed and/or efficiency of cellularreprogramming performed in the absence of the inhibitor.

52. The method of paragraph 50 or 51, wherein the measure of efficiencyof cellular transdifferentiation comprises an increase in the totalnumber of transdifferentiated cells relative to transdifferentiation inthe absence of a said inhibitor.

53. The method of paragraph 50, 51, or 52, wherein the measure of speedof cellular transdifferentiation comprises the appearance oftransdifferentiated cells at an earlier time point than occurs whencells are transdifferentiated in the absence of said inhibitor.

54. The method of any one of paragraphs 50-53, wherein the inhibitorcomprises an RNA interference molecule or an antibody.

55. The method of any one of paragraphs 50-54, wherein the RNAinterference molecule comprises an siRNA or an shRNA.

56. The method of any one of paragraphs 50-55, wherein step (a) isperformed before or during step (b).

57. The method of any one of paragraphs 50-56, wherein thetransdifferentiation of step (b) comprises transdifferentiation of afibroblast to a neuron or transdifferentiation of a B-cell to amacrophage.

58. The method of any one of paragraphs 50-57, whereintransdifferentiation of a fibroblast to a neuron comprisesoverexpression of the transcription factor Ascl1 in a fibroblast.

59. The method of any one of paragraphs 50-58, whereintransdifferentiation of a B-cell to a macrophage comprisesoverexpression of the myeloid transcription factor C/EBPα in a B-cell.

60. The method of any one of paragraphs 50-59, wherein the inhibitor ofthe CAF-1 complex inhibits the Chaf1a and/or Chaf1b subunit of saidcomplex.

EXAMPLES Example 1: Sumo2 and Nutd21 as Roadblocks to Reprogramming

The goal of this study was to identify novel, potent roadblocks toreprogramming by performing a serial genome-wide shRNA enrichment screenin combination with a well-defined transgenic reprogramming system.Unexpectedly, this screening strategy uncovered two post-transcriptionalmechanisms, protein sumoylation and alternative polyadenylation, asstrong repressors of iPSC formation.

Serial shRNA Screen for Roadblocks to Reprogramming

To identify roadblocks to iPSC formation in an unbiased manner, theinventors combined a well-defined transgenic reprogramming system with agenome-wide shRNA library targeting 27,478 genes with 62,877 hairpins.The inventors utilized murine embryonic fibroblasts (MEFs) carrying adoxycycline (dox)-inducible polycistronic cassette encompassing the openreading frames for Oct4, Klf4, Sox2 and c-Myc (OKSM) in the Col1a1locus, the M2-rtTA transactivator in the Rosa26 locus and an EGFPreporter in the endogenous Pou5f1 (Oct4) locus (Stadtfeld et al., 2010).These transgenic MEFs are referred to herein as “reprogrammable cells”and the genotype as “Col1a1-tetOP-OKSM; R26-M2rtTA; Oct4-GFP”. The shRNAlibrary was generated by cloning shRNAs into the pHAGE-Mir lentiviralvector carrying a puromycin resistance gene and an tRFP reporter (Planket al., 2013). Transduction of reprogrammable MEFs with an identicalempty vector gave rise to Oct4-GFP+, tRFP+ iPSC colonies upon exposureto dox, albeit at slightly lower frequencies than uninfected cells(FIGS. 1A, 1B and data not shown), demonstrating the feasibility of ascreen to identify roadblocks to iPSC generation using this lentiviralshRNA vector system.

To identify shRNAs that potently enhance reprogramming with lowbackground signal from passenger shRNAs, pooled screening strategy usingserial enrichment of hairpin libraries was devised. Briefly, theinventors infected reprogrammable MEFs with the pooled shRNA library 2days before dox induction to ensure effective suppression of targetsprior to initiation of reprogramming. After 10 days of OKSM expression,dox was withdrawn for 4 days to select for stably reprogrammed,transgene-independent colonies, followed by purification of emergingOct4-GFP+ cells by flow cytometry (FIG. 1C). Enriched hairpins wereamplified by PCR from genomic DNA and subsequently re-cloned into theoriginal viral backbone before initiating another round of viraltransduction and reprogramming (FIG. 1D). In total, between 3 and 5rounds of shRNA enrichment and iPSC generation were performed (FIG. 1E).Parallel cultures of reprogrammable MEFs were exposed to dox alone ortransduced with the viral library in the absence of dox beforeextracting genomic DNA (FIG. 1C and FIG. 6); these samples served ascontrols for possible passenger hairpins that became passively enrichedin expanding iPSC colonies or hairpins that merely affected the growthof non-induced reprogrammable MEFs. Library representation wasdetermined in all samples by deep (Solexa™) sequencing of genomic DNA.To identify potential hits, the inventors utilized a set of criteriabased on absolute shRNA sequence reads and shRNA enrichment scoresbetween experimental and control samples (FIG. 6).

Nudt21 and Sumo2 Emerge as Top Candidate Roadblocks to Reprogramming

The inventors observed a steep drop in library complexity (i.e., thenumber of unique shRNAs detected by Solexa™ sequencing) after the firstround of reprogramming but a more gradual decline in subsequent rounds(FIG. 1F, left panel). Critically, shRNA libraries prepared from rounds1-3 enhanced the formation of iPSCs compared to uninfected controls,indicating a progressive enrichment of functional hairpins that promotedreprogramming (FIG. 1F, right panel). The inventors next determinedcandidate hairpins that may promote reprogramming based on (i) theirenrichment across all libraries relative to controls and (ii) absoluteshRNA sequence representation per library (FIG. 6). To validatecandidates, the inventors recovered multiple top-scoring shRNAs by PCRfrom enriched shRNA libraries, subcloned hairpins into the pHAGE-Mirvector and infected reprogrammable cells individually with theseconstructs or a control shRNA vector targeting Firefly luciferase. FIG.1G depicts the enrichment of 26 selected candidate shRNAs across 3rounds of screening. Of these shRNAs, 3 hairpins enhanced iPSC formationmore than 2-fold (Nudt21, Eif2a, Izumo4) in initial validationexperiments with Nudt21 showing the strongest effect (FIG. 1H).

To obtain a better coverage of the library and to minimize the loss ofpotentially functional hairpins during the first round of reprogramming,the serial screen was repeated with a higher number of starting cellsand 2 additional rounds of reprogramming and shRNA enrichment. Thismodification of the protocol indeed resulted in a more gradual reductionof library complexity after the first round of reprogramming and aconcomitant enrichment of hundreds of hairpins that enhanced iPSCformation as a pool (FIG. 1I). Analysis of shRNAs that were consistentlyenriched across all 5 rounds of reprogramming using two differentalgorithms revealed several additional candidate barriers toreprogramming such as Fgf5, Dnmt3a, Smpd13a and Sumo2 (FIG. 1J). Of 27validated shRNAs, 7 showed a more than 2-fold increase in iPSC formation(Gstt4, Gm719, Sqrd1, Dnmt3a, BTBD10, Smpd3a, Sumo2) (FIG. 1K) withSumo2 shRNA exhibiting the strongest phenotype. Given the prominenteffects on reprogramming of shRNAs targeting Nudt21 from the firstscreen and Sumo2 from the second screen, the inventors focused on thesegenes for the remainder of the study.

Nudt21 (nucleoside diphosphate linked moiety X-type motif 21; alsotermed Cpsf5 or Cfim25) is part of the cleavage factor involved in 3′RNA cleavage and polyadenylation processing (Di Giammartino et al.,2011) while Sumo2 (small ubiquitin-like modifier 2) plays an importantrole in lysine sumoylation of proteins (Hickey et al., 2012).

Suppression of Nudt21 or Sumo2 Promotes Pluripotency Gene Activation inNascent iPSCs without Compromising Growth or Pluripotency

Using more quantitative reprogramming assays, the inventors found thatsuppression of either Sumo2 or Nudt21 increased the number oftransgene-independent alkaline phosphatase-positive (AP+) iPSC-likecolonies up to 15-fold and the fraction of Oct4-GFP+ cells up to 25-fold(50% with Nudt21 shRNA; 16% with Sumo2 shRNA; 2% with control shRNA atday 8) (FIGS. 2A, 2B). The inventors were able to recapitulate enhancedreprogramming with 4-6 independent shRNAs as well as siRNA poolstargeting Nudt21 and Sumo2, documenting the consistency of the observedphenotype using either permanent or transient knockdown approaches.(FIGS. 2C, 2D and FIG. 7). Importantly, suppression of Sumo2 and Nudt21led to reduced transcript and protein levels of either factor,demonstrating the specificity of knockdown (FIG. 2E, 2F and FIG. 8).Moreover, iPSC generated with these shRNAs could be stably propagatedover many passages and gave rise to well-differentiated teratomas,documenting that suppression of Sumo2 or Nudt21 does not compromise theself-renewal or pluripotency of iPSCs. It is noteworthy that endogenousNudt21 and Sumo2 mRNA levels were comparable between MEFs and iPSCs andbarely changed during the reprogramming process, indicating that thesefactors are important in both somatic and pluripotent cell types (FIG.2G).

To complement the aforementioned marker-based assays of reprogrammingwith a functional assay, it was determined whether suppression of Sumo2or Nudt21 could promote the formation of transgene-independent iPSCcolonies after short pulses of OKSM expression (FIG. 2H). Consistentwith AP and Oct4-GFP-based assays, it was found that knockdown of eithermolecule yielded transgene-independent iPSC colonies after only 5 daysof OKSM expression, whereas stable iPSC colonies only emerged by day 8in controls (FIG. 2I). In agreement with this observation, the inventorsdetected transcriptional upregulation of key ESC-associated genes (e.g.,Epcam, Cdh1 and Sal14) and epigenetic regulators (e.g., Dnmt3b and Tet1)exclusively in cells expressing OKSM and either Nudt21 or Sumo2 shRNAsat day 6 of reprogramming (FIG. 2J). Critically, knockdown of neithermolecule had a discernible effect on cell proliferation or apoptosis ofbulk cultures, thus excluding the possibility that the observedphenotypes are due to accelerated growth or reduced cell death.Together, these results demonstrate that transient or constitutivesuppression of Sumo2 and Nudt21 markedly enhances and accelerates theformation of iPSCs from somatic cells.

Nudt21 and Sumo2 Suppression Act During Early-to-Mid Stages ofReprogramming

In order to understand how Sumo2 and Nudt21 suppression influences thedynamics of iPSC formation, surface markers and a reporter allele wereutilized to distinguish between early, mid and late stages ofreprogramming. The inventors previously showed that cells undergoingsuccessful reprogramming initially upregulate SSEA1 (early stage),followed by sequential activation of EpCAM (mid stage) and Oct4-GFP(late stage) (Polo et al., 2012). Remarkably, Nudt21 suppression showeda noticeable increase in the fraction of SSEA1+ and EpCAM+ cellsrelative to controls as early as day 3 of reprogramming (14% vs. 6% forSSEA1; 19% vs. 1% for EpCAM)(FIGS. 3A, 3B). This trend continued atlater stages of iPSC formation when 56% EpCAM+ cells were detectable byday 5 (8% in controls) and 49% Oct4-GFP+ cells by day 8 (2% incontrols). In contrast, Sumo2 depletion had no pronounced effects on theearliest intermediates of reprogramming, as shown by comparablefractions of SSEA1+ and EpCAM+ cells at day 3 relative to controls (5%vs. 6% for SSEA1; 4% vs. 1% for EpCAM)(FIG. 3A, 3B). However, theinventors observed a 3-fold increase in the fraction of SSEA1+ cells anda 5-fold increase in the fraction of EpCAM+ cells by day 5 as well as an8-fold increase in the fraction of Oct4-GFP+ cells by day 8 ofreprogramming. A relative comparison of SSEA1+, EpCAM+ and Oct4-GFP+cells between virally transduced (tRFP+) and untransduced (tRFP−)reprogrammable cells confirmed these observations and furtherdemonstrated that the enhancing effect of Sumo2 and Nudt21 suppressionon iPSC generation is cell-autonomous (FIG. 3C).

To functionally corroborate the notion that Sumo2 and Nudt21 arerequired during early-to-mid stages of reprogramming, the inventorsdetermined iPSC colony formation efficiencies after transfectingreprogrammable MEFs with siRNAs against Sumo2 or Nudt21 either once (onday 0) or twice (on day 0 and day 3)(FIGS. 3D, 3E). iPSC colonyformation was essentially the same when Sumo2 or Nudt21 were suppressedinitially or continuously during a 6-day reprogramming period.Collectively, these phenotypic and functional assays indicate thatNudt21 suppression promotes very early stages while Sumo2 suppressionpromotes early-to-mid stages of reprogramming, ultimately leading to adramatic increase in the formation of Oct4+ transgene-independent iPSCs.

Nudt21 and Sumo2 Suppression Act Independently of c-Myc Expression andin Parallel with Small Molecule Enhancers of Reprogramming

It was next investigated whether exogenous c-Myc expression was requiredfor the enhancement of iPSC formation by Sumo2 shRNAs and Nudt21 shRNAs,as was previously observed for Mbd3 depletion (Rais et al., 2013). Tothis end, reprogrammable MEFs were derived from mice carrying theCol1a1-tetOP-OKS-mCherry allele (lacking the c-Myc transgene) incombination with the R26-M2rtTA allele (FIG. 4A). Exposure of these MEFsto dox alone gave rise to extremely few, if any, AP+ colonies after 9-21days of OKSM expression, and no Oct4-GFP positive cells could bedetected by day 9 of reprogramming (FIGS. 4B-4D). In stark contrast,depletion of Sumo2 or Nudt21 in these cells using transient transfectionof siRNA pools readily yielded iPSC colonies and stable Oct4-GFP+ cellsby flow cytometry after as little as 9 days of OKSM expression. It wasconcluded that suppression of Nudt21 or Sumo2 enhances reprogrammingindependently of exogenous c-Myc expression, thus enabling iPSCformation from cells under conditions that bypass the use of this potentoncogene.

To determine whether Nudt21 and Sumo2 suppression act in parallel withsmall molecules that were previously shown to enhance reprogramming, theinventors treated reprogrammable cells harboring shRNAs against Sumo2,Nudt21 or Firefly luciferase with doxycycline in the presence or absenceof ascorbic acid (AA)(Esteban et al., 2010), a Dot11 inhibitor(Dot11i)(Onder et al., 2012) and a Gsk3 inhibitor (Gsk3i)(Silva et al.,2008)(FIG. 4E). Consistent with previous reports, it was found thatexposure of reprogrammable cells to each of these compoundssignificantly enhanced the generation of AP+ iPSC colonies, withcombined ascorbate/GSK3 inhibitor treatment (AGi) exhibiting thestrongest effect. Strikingly, Nudt21 suppression alone was as effectiveas AGi treatment while Sumo2 depletion alone even surpassed the effectof AGi on AP+ colony formation (FIG. 4E). Moreover, suppression ofeither Sumo2 or Nudt21 further enhanced iPSC formation in the presenceof ascorbate, Gsk3i or Dot11i (FIG. 4F). These results underscore thestrong effects of individual Nudt21 and Sumo2 suppression on thereprogramming process and indicate that the sumoylation andpolyadenylation pathways may act in parallel to previously describedmediators of iPSC formation including ascorbic acid, H3K79 methylationand Gsk3 signaling.

Generation of iPSCs after as Little as 36-48 Hours of OKSM Expression

Given the additive effect of Nudt21 and Sumo2 suppression with smallmolecule enhancers of reprogramming, the inventors tested whether thiscombination treatment would allow them to further reduce the minimaltime period of OKSM expression required to produce stabletransgene-independent iPSCs. Early passage reprogrammable MEFs (passage2) carrying two copies each of the Col1a1-tetOP-OKSM and R26-M2rtTAalleles were used to achieve optimal reprogramming efficiencies. MEFsexposed to Dot11i, Gsk3i and AA required at least 3 days of OKSMexpression to produce dox-independent AP+ iPSCs, which is faster thanany previously reported protocol. Remarkably, suppression of eitherNudt21 or Sumo2 further reduced this time window to 36 h using Sumo2shRNAs and 48 h using Nudt21 shRNAs (FIGS. 5A-5C). Emerging iPSCcolonies activated the endogenous Oct4-GFP reporter, expressed Nanog andSox2, gave rise to well-differentiated teratomas and supported theformation of coat-color chimeras, indicating that these are authenticiPSCs (FIGS. 5C-5F). These results show that 1-2 days of OKSM expressionare sufficient to produce stable, pluripotent iPSCs when either Nudt21or Sumo2 expression is transiently suppressed.

The inventors identified Sumo2 and Nudt21 as novel roadblocks to iPSCgeneration by combining a well-defined transgenic reprogramming systemwith a genome-wide shRNA screening approach. In contrast to recent shRNAor siRNA screens conducted during iPSC formation, the inventors employeda serial shRNA enrichment strategy, which may reduce the number of falsepositive hits and allow for selection of shRNAs with a strongerphenotype. Indeed, suppression of the top candidates, Sumo2 and Nudt21,markedly enhanced and accelerated iPSC formation compared to individualhits that emerged from previous large-scale screens or candidates thatwere selected based on gene expression differences between somatic andpluripotent cells. In agreement with this notion, the expression ofSumo2 and Nudt21 did not dramatically change during reprogramming. Tothe inventors' knowledge, OKSM expression for 36-48 hours represents theshortest time period that has been reported to generate authentic iPSCsfrom differentiated cells. In addition to Sumo2 and Nudt21, this screenuncovered a number of other candidate barriers to iPSC formation, whichprovide a rich resource for future mechanistic studies of thereprogramming process.

Both Nudt21 and Sumo2 affect post-transcriptional mechanisms, i.e.,alternative polyadenylation (APA)(Di Giammartino et al., 2011) andlysine sumoylation (Hickey et al., 2012), which have not previously beenrecognized as roadblocks to reprogramming. While these data indicatethat both proteins function during early-to-mid stages of reprogrammingin a Myc-independent manner, the precise molecular mechanisms by whicheach factor suppresses iPSC formation remains to be elucidated. Nudt21reportedly suppresses the switch from distal to proximal polyadenylationsites in glioblastoma cells, leading to increased expression oftranscripts involved in cellular proliferation and tumorigenicity(Masamha et al., 2014). Of interest, a global switch from distal toproximal polyadenylation sites has also been observed during cellularreprogramming of MEFs into iPSCs (Ji and Tian, 2009), thus pointing tointriguing parallels between reprogramming and cancer and providing apotential mechanism by which Nudt21 may enhance iPSC generation. Withoutwishing to be bound by theory, the inventors surmise that Nudt21promotes the silencing of somatic transcripts harboring distal APA sitesand the expression of pluripotency-associated transcripts harboringproximal APA sites. It should be informative to follow dynamic changesin APA usage during reprogramming in the presence and absence of Nudt21to test this hypothesis.

The present findings have practical implications for basic science andcell therapy. The ease with which Sumo2 and Nudt21 can be inhibitedusing transient siRNA delivery can facilitate the mechanistic dissectionof the reprogramming process in more homogeneous cell cultures. Theobservation that Sumo2 and Nudt21 depletion cooperate with smallmolecule enhancers of reprogramming but do not require exogenous c-Mycexpression can further facilitate the efficient and safe generation ofpatient-specific iPSCs from rare donor cells.

Methods and Materials Tissue Culture and Virus Production

Reprogrammable Mouse Embryonic Fibroblasts (repMEF) were derived fromday E13.5-15.5 mouse embryos carrying the Col1a1-tetOP-OKSM andRosa26-M2rtTA alleles (Stadtfeld et al., 2010). MEFs with aCol1a1-tetOP-OKS-mCherry allele were used in some experiments (Bar-Nuret al., 2014). Reprogramming was initiated in RepMEFs by adding 20 ng/mldoxycyline (dox) and, where indicated, 25 ug/ml L-Ascorbic acid (Sigma™A4544-25G), 3 uM GSK3 inhibitor (CHIR99021, Stemgent™ or Tocris™), Alk5inhibitor (RepSox, R0158, Sigma-Aldrich™), 1 uM MEK inhibitor(PD0325901, Stemgent™) or Dot11 inhibitor. RepMEFs were typicallyexpanded under hypoxic (4% oxygen) conditions until dox induction. MEFswere infected with shRNA vectors at passage 4 unless noted otherwise,allowed to recover for 48-60 hours, harvested, counted and seeded at adensity of 20K cells per well of a 6-well plate. Media was changed every2 to 3 days. Dox was withdrawn by removing media, washing with 1×PBS,and continuing culture in ESC media (Knockout DMEM, 1,000 U/ml LIF, 20%FBS). For phage or GipZ virus preparation, 293T cells were seeded andtransfected at 50% confluency with PEI (Polyethylenimine) and DNA(vector+psPax2 and pDM2.G) at a ratio of 3:1 in Optimem™ media (LifeTechnologies™). After 24 hours, supernatant was collected through a 0.4um filter and mixed 3:1 with fresh MEF media and polybrene (10 ug/ml)for direct infection or precipitated with PEG (Polyethylglycol) and keptat −80 C for later infections. Viral transductions were performed byspin infection for 30 minutes at 2,150 rpm at room temperature. Freshmedia was added ˜16 hours after infections.

siRNA Transfections

Transfections of RepMEFs with siRNAs were performed in 12-well plates atthe time of dox administration. Per 12-well plate, 2 ul ofLipofectamine™ 2000 was added to 75 ul of Optimem™ (Life Technologies™)and 1.5 ul of siRNA (esiRNA, Sigma™) was added to 75 ul of Optimem™;both mixtures were separately incubated at room temperature for 5minutes, then combined and incubated for another 15 minutes beforeadding the solution to the reprogramming media (normal media lackingantibiotics). After overnight incubation, the transfection media wasreplaced with regular reprogramming media.

Flow Cytometry

To determine viral infection efficiency and cell numbers beforeinitiation of reprogramming, infected MEFs were harvested using 0.25%trypsin-EDTA (Life Technologies™) and kept at 4° C. A small aliquot wasanalyzed on the MACSQuant cytometer to determine the number of cells/mland the percentage of tRFP+ cells. To prepare growth curves, 3replicates were harvested at each time point to measure cell counts(DAPI-negative live cells). Intermediates of reprogramming were analyzedby staining with Thy1-Viogreen (BD™), SSEA1-APC (Biolegend™),SSEA1-PE-Cy7 (Miltenyi Biotec™), and/or Epcam-PE-Cy7 (eBioscience™)(1:200 for 30 min at 4° C.). To measure the fraction of dying cells,BD™'s Annexin V kit was used according to manufacturers' instructions.All cytometry data was analyzed and plotted using FlowJo software.Fluorescence-activated cell sorting (FACS) was performed by harvestingcells with Trypsin-EDTA (Gibco™) before resuspending cells in MEF mediaand subsequent filtration through 100 um and 40 um filters. Forisolation of Oct4-GFP+ iPSCs, SSEA1+ cells were enriched by labelingwith SSEA1 antibody coated magnetic beads and MACS sorting. The positivefraction was then purified for Oct4-GFP+ cells by FACS. For analyses ofpre-MACS and post-MACS samples, aliquots were stained using Thy1-PacificBlue (eBioscience™) and SSEA1-APC (Biolegend™) antibodies, 1:200 in 1%FBS:PBS, 30 min at 4° C. Analysis was done using the FACS Diva software.

Cell Cycle Analysis

To determine cell cycle dynamics, repMEFs treated with dox for 48 h wereexposed to 20 uM BrdU (Sigma™) for 30 minutes in regular media,trypsinized and kept on ice in 100 ul 1×PBS. To fix cells, 2 ml of coldEtOH was added dropwise, incubated for 30 minutes, followed by additionof 2 ml 4N HCl and another 30 minutes of incubation. Cells were thenspun down at 500 rpm for 5 min. at 4 C and resuspended in 1 ml of 0.1NNa₂B₄O₇, pH 8.5 and washed with staining buffer (2% FBS and 0.5% Tween20 in PBS). Antibody for BrdU (mouse, DAKO M074401-8) was added to cellsfor 30 min at room temperature (RT) at a concentration of 5 ug/ml,followed by 3 washes in 1×PBS and incubation with anti-mouse FITCsecondary antibody (BD Biosciences™, 55434) for 30 min at RT at adilution of 1:100. After 3 additional washes with 1×PBS, the pellet wasresuspended for analysis in 2% FBS/PBS containing 5 ug/ml propidiumiodide. Resuspension volume was used to normalize for cell count of eachsample. Samples were analyzed immediately on the MACSQuant cytometer.

Quantification of Reprogramming Efficiencies

For macroscopic detection of iPSC colonies, Alkaline Phosphatase (AP)staining was carried out according to manufacturer's instructions usingthe Vector Labs™ AP staining kit (#5100). AP staining was alwaysperformed 2 to 4 days after dox withdrawal to eliminate partiallyreprogrammed colonies and score for transgene independent iPSCs.Colonies were counted manually or by custom-made Nikon™ software(CL-Quant™).

Teratoma and Chimera Formation

For teratoma generation, iPSC lines (passage 6 or higher) were harvestedand resuspended in 600 ul media per confluent 6-well. Mice wereanesthetized with Avertin and injected with 150 ul cell suspensionsubcutaneously. Tumors were harvested 3 to 4 weeks after injection andanalyzed histologically. For chimera production, iPSC lines wereinjected as single cell suspension into day 3.5 blastocysts isolatedfrom intercrosses of C57Bl/6xBDF1 mice. Blastocysts were transferredinto pseudo-pregnant Swiss Webster recipient animals.

Immunofluorescence Analysis

iPSC lines (passage 6 or higher) were seeded in a 24-well plate at a lowdensity. Once small colonies emerged, wells were washed with 1×PBS andfixed by a 5-10 minute incubation in 10% formalin at room temperature.After washes in 1×PBS, cells were blocked in 1×PBS containing 2% BSA and0.1% Triton-X 100. Primary and secondary antibodies were diluted inblocking solution at a concentration of 1:200 and added for 1 hour at RTor overnight at 4 C. Primary antibodies were anti-mouse Nanog (Abcam™),Sox2 and Oct4 (Santa Cruz™); secondary antibodies were donkey anti-goatIgG or donkey anti-rabbit IgG Alexa Fluor 546-conjugated antibodies(Life Technologies™) After 2 washes in 1×PBS, cells were immobilized onslides in mounting media containing DAPI (Vectashield™, Vector Labs™)and analyzed.

RNA Expression Analysis

Total RNA was isolated from repMEFs in triplicates exposed to dox for 3or 6 days using the QIAGEN™ RNeasy™ Mini kit and sequencing librarieswere prepared as described (Shepard et al., 2011) and sequenced at theTufts University Genomics core. Activation of pluripotency-associatedgenes at day 6 of reprogramming was determined based on these expressiondata. For quantitative PCR analysis, Brilliant III SYBR-green basedmaster mix was used according to the manual (Agilent™), following RNAisolation (RNeasy™ kit, QIAGEN™) and reverse transcription (TranscriptorFirst Strand cDNA Synthesis Kit, Roche™) of Nudt21, Sumo2, or controlknockdown samples two days after initiation of reprogramming. Sampleswere run in triplicate on the Lightcycler 480 (Roche™).

Western Blot Analysis

Protein lysates were run on 4-20% Mini Protean TGX gels (Bio-Rad™),blotted onto Immobilon-P membrane (EMD Millipore™) and stained withanti-Nudt21 (Santa Cruz™), anti-Sumo2 (Life Technologies™) or anti-Actin(Abcam™) antibodies.

shRNA Screen and Hits Identification

RepMEFs were expanded until passage 4 in 4% oxygen, switched toatmospheric oxygen, and infected with the pooled shRNA library asdescribed above. For each shRNA (621,000 shRNAs in total), 1-2×10³ cellswere infected to achieve good coverage. Infected cells were passagedonto gelatinized 15 cm cell culture dishes (Falcon™) in reprogrammingmedia (ESC media supplemented with ascorbic acid and doxycycline) for 10days, and in doxycycline/ascorbic acid-free ESC media for an additional4 days. Cells were harvested, pooled, and purified with SSEA1-linkedmagnetic beads using an AutoMACS sorter (Miltenyi™). SSEA1-enrichedcells were then FACS-sorted for endogenous Oct4-GFP expression.

Genomic DNA was extracted from collected Oct4-GFP+ cells by lysing thecells in 10 mM Tris pH 8.0, 10 mM EDTA, 10 mM NaCl, 0.5% Sarkosyl.Lysates were treated with 0.1 mg/ml Rnase A at 37° C. for 30 min, 0.5mg/ml Proteinase K at 55° C. for 1-2 hr, and then phenol chloroformextracted, ethanol precipitated, and resuspended in 10 mM Tris-HCl pH8.0. For each sample, all of the genomic DNA was used as template forshRNA PCR, usually in multiple PCR reactions. Each 50 μl PCR reactioncontained: 2.5 μg genomic DNA template, 200 uM dNTPs, 400 nM of each PCRprimer (pHAGE-Mir-PCR: 5′-GCAAACTGGGGCACAGATGATGCGG; BC1R L:5′-CGCCTCCCCTACCCGGTAGA), 1× Q5 reaction buffer, 1× Q5 high GC buffer,and 0.5 μl Q5 polymerase (NEB™). PCR was performed with the followingprogram: 94° C. 4 min, 35 cycles of (94° C. 30 sec, 60° C. 30 sec, 72°C. 45 sec), 72° C. 10 min. PCR products (˜700 bp) for each sample werepooled, ethanol precipitated, resuspended, and gel-purified using theQIAquick™ Gel Extraction Kit (Qiagen™). The purified shRNA PCR productswere used to: 1) generate sublibraries for the next round of shRNAlibrary screens; 2) generate sequencing libraries for Solexa sequencing.

For sub-library generation, the purified PCR product was digested withNotI and MluI, and the ˜400 bp fragment that contains the shRNAs wasgel-purified. Separately, the pHAGE-Mir plasmid was also digested withNotI and MluI to recover the ˜9 kb vector backbone. 25-50 ng of thepurified shRNA fragment and 125-250 ng of the vector backbone wereligated in 5 ul ligation reaction using NEB™ T4 ligase. 1 ul ligationreaction was used to transform 20 ul Electromax competent cells DH10b(Life Technology™) with electroporation. 1 ul of the transformationreaction was plated on one 10-cm LB-Amp (100 ug/ml) plate to estimatethe total number of colonies, and the rest of the transformationreaction was plated on two 15-cm LB-Carbenicillin (100 ug/ml) plates andgrown overnight at 37° C. To maintain the representation of the library,at least 100× coverage was needed (i.e., colony number=100×number ofshRNAs in the library). When necessary, the entire ligation reaction maybe used for transformation in multiple electroporation reactions toincrease the number of colonies. The next day, lawn formed on the two15-cm plates were scraped off and cultured in 300 ml LB-Carbenicillin(100 ug/ml) medium and grown at 30° C. for 2-3 hrs. The bacteria wascollected and the cloned sub-library DNA was extracted by the Genelute™Maxiprep kit (Sigma™).

For Solexa sequencing, the purified shRNA PCR product was used astemplate for another round of PCR: 500 ng purified shRNA PCR product,200 uM dNTPs, 2 uM of each PCR primer (p5 and p7), 1× Q5 reactionbuffer, 1× Q5 high GC buffer, and 1 μl Q5 polymerase (NEB™) in 100 ulPCR reaction. PCR was performed with the following program: 94° C. 4min, 2 cycles of (94° C. 30 sec, 50° C. 20 sec, 72° C. 30 sec), 20cycles of (94° C. 30 sec, 60° C. 20 sec, 72° C. 30 sec), 72° C. 10 min.PCR products (˜120 bp) were and gel-purified using the QIAquick™ GelExtraction Kit (Qiagen™). Gel-purified products were submitted forSolexa™ sequencing on the Illumina™ MiSeq instrument, using a customsequencing primer: mir30-EcoRI: 5′-TAGCCCCTTGAATTCCGAGGCAGTAGGCA

PCR Primers:

p5-miSeq: 5′-ATGATACGGCGACCACCGAGATCTACACCTAAAGTAGCCCCTTGAAT TC;p7-miSeq-1: 5′-CAAGCAGAAGACGGCATACGAGACGATAGTGAAGCCACAGATGTA p7-miSeq-2:5′-CAAGCAGAAGACGGCATACGAGACACTAGTGAAGCCACAGATGTA p7-miSeq-3:5′-CAAGCAGAAGACGGCATACGAGACTATAGTGAAGCCACAGATGTA p7-miSeq-4:5′-CAAGCAGAAGACGGCATACGAGACCTTAGTGAAGCCACAGATGTA

Different p7 primers were used for multiplexing purpose.

Single-end 51 bp reads were obtained using the Illumina HiSeq or MiSeqinstrument. The reads are expected to have an initial 22 nucleotidesthat identify the shRNA, followed by a constant region that is the samefor all shRNAs and a 2 nucleotide barcode to identify the sample. Readsthat contain perfect matches at the following 6 nucleotides were firstextracted from the sequencing data: the 2 nucleotides adjacent to theinitial 22 base sequence and the 2 nucleotides adjacent to the barcodeon both sides. The shRNAs were then identified by requiring an exactmatch of the 22 nucleotides to the sequences in the shRNA libraryannotation file. The samples were identified by the 2 nucleotidebarcodes.

The total number of reads that were identified for each shRNA, sample,and round were counted. The counts were normalized to be directlycomparable between samples and rounds by first dividing by the totalnumber of counts for that sample and round and then multiplying by thetotal number of shRNAs in the initial library. A pseudocount of 1 wasadded to each normalized count to downweight enrichment derived from lowread counts and to avoid division by zero in calculating fold-changes.

The enrichment for each shRNA in each round was calculated as the log 2fold change of the Oct4-GFP+ normalized counts over the maximum of thenormalized counts of the controls (TO, No-Dox, and Oct4-GFP−). Thecumulative enrichment for each shRNA in each round was calculated as thesum of the log 2 fold changes for that round and all previous rounds.The overall enrichment of each shRNA was defined as the maximum of thecumulative enrichment scores among all rounds.

The heat map for FIG. 1J was plotted using the cumulative enrichmentscores. Only shRNAs that have at least one read in the Oct4-GFP+ samplein at least two rounds were used in the plot, resulting a total of23,853 shRNAs.

TABLE 1 List of Primers Target Purpose Forward oligo Reverse OligoNudt21 qPCR CGGCTTCTTTTACTTC GGGGTATGGACCCATC TGCATAC ATTT Sumo2 qPCRAAGGAAGGAGTCAAGACT CGGAATCTGATCTGCC GAGAA TCATTG GAPDH qPCRAGG TCG GTG TGA TGT AGA CCA TGT AGT ACG GAT TTG TGA GGT CA phage vectorrecloning shRNA PCR5-3: BC1R: GCAAACTGGGGCACA CGCCTCCCCTACCCGGGATGATGCGG TAGA phage vector sequencing TAGCCCCTTGAATTCC N/A shRNAGAGGCAGTAGGCA phage vector half hairpin JH353F: BC1R: amplification forTAGTGAAGCCACAGA CGCCTCCCCTACCCGG sequencing library TGTA TAGAhalf hairpin adaptor addition P5 + mir3: P7 + loop: amplicon for SolexaAATGATACGGCGACC Barcode xx = GA, TG or sequencing ACCGACTAAAGTAGC AT:CCCTTGAATTC CAAGCAGAAGACGGCA TACGAxxTAGTGAAGCC ACAGATGTA

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Example 2: The Histone Chaperone CAF-1 Safeguards Somatic Cell IdentityDuring Transcription Factor-Induced Reprogramming

The generation of iPSCs from somatic cells upon forced expression of thetranscription factors Oct4, Klf4, Sox2 and c-Myc (OKSM) is a highlydynamic and lengthy process that proceeds through heterogeneousintermediate cell populations, which poses challenges for genetic andbiochemical dissection of the underlying mechanisms (9,10). Previousefforts to identify chromatin modulators of iPSC formation included gainand loss of function screens as well as transcriptional profiling ofbulk or FACS-enriched cell populations undergoing reprogramming. Whileinformative, these approaches remain limited in several ways. Forexample, iPSC modulators that do not change transcriptionally aretypically overlooked when analyzing expression dynamics in reprogrammingintermediates (11). Moreover, known repressors of iPSC formation such asp53, Mbd3, Dot11, and Dnmt1 were either predicted or identified fromsmall sets of candidate gene lists, leaving open the possibility thatmajor roadblocks to reprogramming remain to be discovered (12-15).Genome-wide RNAi screens are challenging due to various technicallimitations such as lack of effective shRNAs when expressed from asingle genomic copy, prevalent off-target effects, as well as biases inthe library representation or the screening readout (11, 16, 17).Indeed, previous RNAi screens identified a number of chromatinregulators of iPSC formation with little to no overlap among independentstudies. Furthermore, certain chromatin regulators reportedly haveopposing effects on iPSC generation when tested in different cellularcontexts and culture conditions, indicating that they may not act asuniversal barriers to reprogramming (14, 18-23). Although thesefunctional and molecular analyses of iPSC formation provided importantinsights into transcription factor-induced reprogramming, the cellularand molecular mechanisms inherent to the induction of pluripotency andtheir parallels to other types of cell fate change remain largelyelusive (24,25).

To systematically explore chromatin factors involved in the maintenanceof somatic cell identity, the inventors assembled custom-designedmicroRNA-based shRNA libraries targeting known and predicted chromatinregulators. 243 known chromatin regulators were used in an arrayedscreening approach during the reprogramming of fibroblasts into iPSCs(26). This screen validated previously implicated chromatin pathways andrevealed novel, more potent repressors of reprogramming. Through aseries of cellular and molecular studies, the inventors demonstrate thatsuppression of a histone chaperone complex most dramatically enhancesand accelerates iPSC reprogramming as well as other cell fatetransitions. It is proposed that this complex functions as a keydeterminant of cell identity by influencing chromatin structure andtranscription factor binding.

Chromatin Focused shRNAmir Screens Systematically Identify GlobalSuppressors of Reprogramming.

To explore chromatin barriers to induced pluripotency in a systematicand comprehensive manner, the inventors used a chromatin-focusedscreening method to identify microRNA-based shRNA (shRNAmir) librariesin a highly standardized reprogramming assay. Specifically, theyutilized transgenic (“reprogrammable”) mouse embryonic fibroblasts (MEF)harboring a doxycycline (dox)-inducible polycistronic OKSM cassette anda constitutive M2-rtTA driver (26). By ensuring controllable andhomogeneous factor expression, this system enables the generation ofstable, transgene-independent iPSCs with reproducible kinetics andfrequencies. The screening platform further provides a means to studychanges in the temporal requirement for OKSM overexpression whilesuppressing candidate chromatin barriers during induced pluripotency.

An arrayed screening strategy was designed using a previously describedmiR30-based shRNA library targeting 243 known chromatin regulators (27)(FIG. 12A). A total of 1,071 experimental and four control shRNAmirswere cloned into a constitutive retroviral expression vector (pLMN) andtransduced one-by-one into reprogrammable MEFs (FIG. 10A). Cells wereinduced to reprogram by addition of dox, followed by a period of doxwithdrawal to select for transgene-independent iPSC colonies. Alkalinephosphatase positive (AP+) iPSC-like colonies were quantified usingcustomized image analysis software, and reprogramming efficiency ratioswere calculated relative to a control shRNA targeting Renilla luciferase(Ren.713).

Nucleosome Assembly as a Major Roadblock to iPSC Formation

The screen identified shRNAs that strongly and consistently promotediPSC formation. The most prominent hits that emerged from the screenwere Chaf1a and Chaf1b, two subunits of the chromatin assembly factorcomplex CAF-1. Among the 22 shRNAs that enhanced iPSC formation morethan four-fold in the arrayed screen, the inventors identified sixshRNAs targeting Chaf1a or Chaf1b (three shRNAs each) including thethree top-scoring shRNAs overall (FIG. 10B, 11A). Importantly, alltested top-scoring shRNAs targeting Chaf1a and Chaf1b reduced theexpression of their predicted target genes (FIGS. 15A, 15B).

Suppression of CAF-1 Accelerates iPSC Formation

To gain insights into the dynamics of reprogramming in the absence ofthe identified chromatin barriers, the inventors followed the emergenceof Epcam+ (early programming marker) and Oct4-tomato (late reprogrammingmarker) cells over time. This showed that Chaf1a suppression increasedthe fraction of both Epcam+ and Oct4-tomato+ cells at day four and sixof reprogramming (FIG. 11B).

To complement these reporter and marker-based assays with a functionalreadout, the inventors examined the ability of candidate hairpins tofacilitate transgene-independent clonal growth, a hallmark of authenticiPSCs. Suppression of Chaf1a indeed gave rise to transgene-independentAP+ cells after as little as four days of OSKM expression when no iPSCswere yet detectable in control shRNA-treated cells (FIG. 11C). Based onthe identification of Chaf1a and Chaf1b as the top hits in twoindependent chromatin-focused reprogramming screens and their dramaticeffects on both the dynamics and efficiency of iPSC formation, furtheranalyses were focused on these two components of the CAF-1 complex.

It was ensured that suppression of CAF-1 subunits does not significantlyinfluence expression of the Col1a1::tetOP-OKSM; R26-M2rtTA system at theRNA or protein level, ruling out the possibility that the observedphenotype is due to direct modulation of the reprogramming transgenes.Moreover, the reprogramming increase elicited by Chaf1b shRNAs could berescued by overexpression of an shRNA-resistant version of human CHAF1BcDNA, demonstrating specificity of the effect. Lastly, knockdown ofeither CAF-1 subunit did not increase cell proliferation rates in thepresence of OKSM induction, thus excluding that the dramatic increase inOct4-GFP+ cells upon CAF-1 suppression is simply due to an expansion ofreprogrammed cells or a loss of unreprogrammed cells.

Reprogramming Phenotype Depends on Optimal CAF-1 and OKSM Dose

To determine whether CAF-1's effect on reprogramming depends on OKSMexpression levels, iPSC formation efficiencies between reprogrammableMEFs carrying either one or two copies of the Col1a1::tetOP-OKSM andR26-M2rtTA alleles were compared; it was previously shown thatincreasing the dose of OKSM and M2-rtTA using this transgenic systemprofoundly influences reprogramming efficiency and speed^(6,16). WhileCAF-1 suppression in heterozygous MEFs increased iPSC formation byorders of magnitude, CAF-1 suppression in homozygous MEFs resulted in amore modest increase in iPSC numbers (FIG. 12A, 12B). Consistently, itwas observed that CAF-1 knockdown had a much stronger effect onreprogramming efficiency when infecting MEFs with viral vectorsachieving either temporally restricted or moderate OKSM expressioncompared to vectors achieving high OKSM expression levels over prolongedperiods of time Enhanced iPSC formation upon CAF-1 knockdown was furtherobserved with an independent transgenic reprogramming system producingOKSM at different stoichiometries compared to our Col1a1::tetOP-OKSMcassette³⁰. These results show that the reprogramming phenotype uponCAF-1 suppression is influenced by both the levels and the duration ofOKSM expression.

CAF-1 is essential for embryonic growth and the viability of culturedcells^(27-29,31). In line with this observation, it was found that thetop-scoring shRNAs targeting CAF-1 components compromised the growth ofNIH3T3 cells in the absence of OKSM expression. To test whether theduration and degree of CAF-1 suppression also affects reprogrammingefficiency, transgenic MEFs carrying a dox-inducible Chaf1a shRNA linkedto an RFP reporter in the Col1a1 locus were generated. When exposingthese cells to either low or high concentrations of dox, a concomitantchange in RFP reporter signal and Chaf1a protein levels (data not shown)was observed. Infection of transgenic MEFs with a constitutivelentiviral vector expressing OKSM in the presence of low doses of doxfor 2, 4, 6, 8 or 9 days resulted in a progressive increase in theformation of Nanog+ iPSC colonies, which plateaued by day 6. Incontrast, exposure of replicate cultures to high doses of dox increasedreprogramming efficiency until day 4 but decreased iPSC colony numbersthereafter. Collectively, these data indicate that enhancedreprogramming is also dependent on CAF-1 dose, with early CAF-1suppression being beneficial but long term, potent suppression beingdetrimental to iPSC derivation.

CAF-1 Depletion Enhances Reprogramming and Direct Lineage Conversion ofDifferent Cell Types

To investigate whether CAF-1 acts as a gatekeeper of cellular identityacross different cell types, the inventors expanded their analysis fromfibroblasts to blood progenitors. To this end, the inventors tested theeffect of Chaf1a knockdown on the reprogramming potential ofhematopoietic stem and progenitors cells (HSPCs) isolated from fetallivers of reprogrammable mice (FIG. 13A). The inventors monitoredexpression of the surface molecule Pecam over time, which coincides withactivation of the Oct4-GFP reporter during reprogramming (10). Afterfour days of OKSM induction, Pecam expression was detectable in 56% ofcontrol HSPCs, while Chaf1a suppression by two independent shRNAsenhanced this fraction to over 90% (FIG. 13B, 13C). By day six,essentially all Chaf1a shRNA treated cells (97%) had completedreprogramming as judged by Pecam surface expression, whereas only 76% ofcontrol cells had acquired a Pecam+ iPSC-like state. Consistently,suppression of Chaf1a gave rise to more transgene-independent coloniescompared to controls at each examined time point. Hence, CAF-1 alsofunctions as a barrier to iPSC formation in a cell type that is lessdifferentiated and intrinsically more amenable to reprogramming comparedto MEFs.

To assess whether CAF-1 stabilizes somatic cell identity in cellfate-conversion systems other than OKSM-mediated reprogramming, theinventors first examined the transdifferentiation of fibroblasts intoinduced neuronal (iN) cells upon overexpression of the transcriptionfactor Ascl1 in MEFs (40). The inventors transduced transgenic MEFsharboring dox-inducible shRNAs targeting Renilla luciferase or Chaf1awith a dox-inducible Ascl1-expressing lentivirus and measured iNformation at day 13. CAF-1 knockdown consistently resulted in a two-foldincrease (P=0.003) in the number of Map2+ neurons in four independentexperiments (FIGS. 13E, 13F).

The inventors next tested the effect of CAF-1 suppression during theconversion of pre-B cells into macrophages upon overexpression of themyeloid transcription factor C/EBPα (FIG. 13G). Exposure of aC/EBPα-inducible pre-B cell line to estradiol reportedly triggersconversion into macrophages within 48 hours at 100% efficiency (41).Remarkably, knockdown of CAF-1 in this cell line resulted in an up tofive-fold increase in the expression of the myeloid markers Cd14 andMac1 after as little as 24 hours of estradiol treatment (FIG. 13H, 13I).Although the fractions of Cd14+ and Mac1+ cells were comparable betweenCAF-1 shRNA and control shRNA conditions at 48 hours, the expressionlevels of both differentiation markers were noticeably higher in CAF-1depleted cells (FIG. 13H). Taken together, these data demonstrate thatCAF-1 suppression not only enhances the induction of pluripotency fromdifferent cell types but also facilitates cellular transdifferentiation,indicating that reduced expression of CAF-1 generally promotes cellularplasticity during transcription factor-induced cell fate conversions.

Depletion of CAF-1 Promotes Chromatin Accessibility at Enhancer Elementsand Facilitates Transcription Factor Binding

Since CAF-1 functions as a histone chaperone that assembles nascentnucleosomes during DNA replication (42), it was reasoned that itsreduced levels may result in a more accessible chromatin structure andthus facilitate transcription factor binding to their target loci. Tointerrogate possible differences in chromatin structure between CAF-1depleted and control cells undergoing reprogramming, the inventors firstperformed SONO-Seq (43) analysis, which allows mapping of accessiblechromatin regions due to their increased susceptibility to sonication.The inventors analyzed bulk cultures expressing OKSM for three days whenno stable iPSCs are yet present. Given the importance of regulatoryelements in defining cell identity (44), the study focused analysis onall annotated ESC-specific promoter and enhancer regions, as defined byrecent ChIP-Seq analyses of post-translational histone modifications andp300 occupancy (45). Notably, while promoter elements showed nodiscernible difference in accessibility (P value=0.51), the inventorsobserved a significant enrichment of SONO-seq signal at ESC-specificenhancer elements in CAF-1 depleted cells at day three of OKSMexpression (value <2.64×10⁻¹²). This observation indicates that CAF-1suppression results in a more accessible local chromatin environmentover ESC-specific enhancer elements early in reprogramming.

To validate these observations and generate a higher resolution map ofchromatin accessibility in early reprogramming intermediates, theinventors performed an assay for transposase accessible chromatin usingsequencing (ATAC-seq), which detects integrations of the Tn5-taggedtransposase in open chromatin regions (46). In agreement with theSONO-seq data, ATAC-Seq analysis of early reprogramming intermediatesshow a more accessible chromatin configuration at ESC-specific enhancersupon CAF-1 depletion compared to controls (P value <3.61×10⁻⁵). Theinventors next interrogated “super-enhancers”, a recently describedclass of major lineage-specific regulatory elements in ESCs and othercell types (47-49). CAF-1 knockdown caused a significant increase inchromatin accessibility when considering all 231 reported ESC-specificsuper-enhancers compared to controls at day three of iPSC formation (Pvalue <1.28×10⁻⁵). This difference became much more pronounced at daysix of reprogramming, consistent with transitioning of the cells towardsa pluripotent state.

When focusing on individual super-enhancers, it was observed that someloci (e.g., Sox2 and Sal14; 42 super-enhancers in total) but not otherswere significantly more accessible by ATAC-seq analysis in CAF-1depleted cells compared to controls. Taken together, these results showthat CAF-1 suppression facilitates a more accessible local chromatinstructure at pluripotency-specific enhancer elements, includingsuper-enhancers. Lastly, the inventors performed ChIP-Seq analysis forSox2 at day three of OKSM expression in order to test the hypothesisthat increased chromatin accessibility at enhancer elements influencesreprogramming factor binding. Indeed, the inventors detected asignificant increase in Sox2 binding to ESC-specific regulatory elementsin CAF-1 knockdown cells compared to controls (FIG. 13C; P value<2.2e⁻¹⁶). While the majority of Sox2 binding sites (around 90%) wereshared between CAF-1 knockdown and control cells, roughly 10% weresignificantly enriched in cells expressing either CAF-1 shRNA (1,329peaks) or Renilla shRNA (1,806 peaks) (FIG. 13D, left panel).Remarkably, binding sites unique to CAF-1 depleted cells were stronglyenriched for ESC-specific Sox2 targets (50) relative to control cells(FIG. 13D, right panel). Consistently, ESC-specific super-enhancerelements were three-fold more abundant among the unique Sox2 sites inCAF-1 knockdown cells compared to controls (15 vs. 5). Of the 15Sox2-bound super-enhancers unique to CAF-1 knockdown cells, seven (47%)also showed a more accessible chromatin structure by ATAC-Seq analysis(e.g., Sal11 and Mycn loci; FIG. 13E). Collectively, these resultsindicate that loss of CAF-1 contributes to reprogramming, at least inpart, by increasing chromatin accessibility at pluripotency-specificenhancer elements and by promoting binding of reprogrammingtranscription factors such as Sox2 to its target genes.

CAF-1 Suppression Alters Local Heterochromatin Domains and PrimesPluripotency Genes for Transcriptional Activation

Considering that CAF-1 plays crucial roles not only in histone exchangebut also heterochromatin maintenance^(27,31,44), the global distributionof the heterochromatin mark H3K9me3 during reprogramming was examined.Significant differences were not detected in H3K9me3 levels acrosspluripotency-associated enhancers or transposable elements, whichrepresent abundant and prototypical heterochromatic regions. Likewise,RNA-Seq analysis of the same intermediates failed to show differentialexpression of transposable elements between control and CAF-1 knockdowncells throughout the reprogramming time course. However, a localdepletion of H3K9 trimethylation was detected at a subset of somaticheterochromatin areas termed “reprogramming-resistant regions”, whichhave recently been linked to the low efficiency of somatic cell nucleartransfer⁴⁵ (FIG. 14D). It is inferred from these data that CAF-1inhibition, in concert with OKSM expression, causes local changes inthis key repressive histone modification, which may prime chromatinstructure for efficient transcriptional activation.

Finally, it was investigated whether the observed CAF-1 shRNA-inducedchromatin changes affect gene expression of associated genes. Of note,no major gene expression differences were detectable between control andCAF-1 shRNA samples at day three of reprogramming (data not shown).However, a subset of genes with increased chromatin accessibility at daythree was transcriptionally upregulated by day six of reprogramming inCAF-1 knockdown intermediates (e.g., Utf1, Epcam, NrOb1, Tdgf1, Sa114),supporting the view that CAF-1 suppression primes the genome forsubsequent transcriptional activation. Altogether, these genome-wideassays support the conclusion that CAF-1 suppression, in collaborationwith potent coactivators, facilitates the transcriptional activation ofsomatically silenced genes.

The inventors have combined a highly standardized iPSC reprogrammingassay and two innovative shRNA screening approaches to systematicallyexplore chromatin barriers to cellular reprogramming. Remarkably, themost potent roadblocks emerging from these screens was the nucleosomeassembly complex CAF-1, which has not been identified in previousgenome-wide or focused screens for iPSC reprogramming barriers (11, 13,14, 16, 17). Of note, CAF-1 and several other roadblocks uncovered inour screens (e.g. Brd4, Dnmt1) are essential genes (51-56), that wouldnot have been identified with alternative screening methods involvingthe complete and permanent ablation of gene function.

Together, these findings highlight the advantage of RNAi technology forgenerating hypomorphic states of gene function in order to dissect basiccellular processes with a comprehensive functional genetics approach.

In addition to identifying CAF-1 as the most prominent roadblock to iPSCformation in two independent screens, this study provides evidence thatCAF-1 suppression facilitates transcription factor-driven cell fatechanges in at least two additional systems: direct conversion offibroblasts to neurons and B cells to macrophages. Consistent with therole of CAF-1 in replication-coupled nucleosome assembly, it was foundthat its suppression has a more pronounced effect on cellular plasticityin systems that involve multiple rounds of cell division (i.e., iPSCformation from MEFs) compared to those that require only 1-3 divisions(i.e., direct lineage conversion) or those that exhibit intrinsicallyhigh proliferation rates (i.e., iPSC induction from HSPCs). Based onthese observations, it is tempting to speculate that a tolerablereduction in the expression of other components associated with the DNAreplication machinery may also facilitate cell fate change. In supportof this idea, hypomorphic alleles of the essential proliferating cellnuclear antigen (PCNA) gene, which acts as a scaffold for proteinsinvolved in DNA replication, suppress position effect variegation inflies (57). The methyltransferase Dnmt1 represents another forkcomponent that is involved in the maintenance of cell identity (3) andscores in the present screen as a chromatin barrier. Although knockdownor pharmacological inhibition of Dnmt1 also facilitates iPSC formationand transdifferentiation of MEFs into myogenic cells (12, 58, 59), thescreens described herein indicate that interference with nucleosomeassembly may represent a more potent and broadly applicable strategy tofacilitate cell fate change.

Without wishing to be bound by theory, the inventors propose that CAF-1contributes to the maintenance of somatic cell identity in replicativecells by safeguarding chromatin structure. According to this model,suppression of CAF-1 triggers dilution of newly assembled nucleosomes atkey enhancer elements and loosening of chromatin structure inconjunction with forced expression of lineage-specifying transcriptionfactors. These combined changes may generate an accessible chromatinlandscape for efficient transcription factor binding and subsequentrobust activation of key target genes. Other histone chaperones maycompensate for the loss of CAF-1 during reprogramming.

Indeed, suppression of the CAF-1 subunit p60 in human cells triggerscompensatory deposition of the histone variant H3.3 by the chaperoneHIRA (60,61). Of interest, H3.3 deposition on chromatin has recentlybeen associated with enhanced reprogramming in the context of somaticcell nuclear transfer (62-64). Given that CAF-1 associates withregulators of heterochromatin, it will further be interesting toascertain whether its suppression during iPSC formation also influencesheterochromatin domains, which are thought to resist reprogramming(29-31, 65).

The present study provides fundamental insight into chromatin-associatedmechanisms regulating somatic cell identity, and indicates novelstrategies for controlling cell fate transitions for therapeuticpurposes. For example, the inventors anticipate that some of thechromatin barriers to iPSC formation identified here also contribute tonormal development and tumorigenesis. Targeting of these chromatinfactors in diseased or damaged tissues can thus help to eliminateaberrant cells or promote cellular regeneration, respectively. The dosedependency of the top hits indicates that one can accomplish this goalwith inhibitors that trigger such cell fate changes in vitro or in vivowith low cellular toxicity.

Methods and Materials Cell Culture and Media

Packaging cells (Platinum-E™ Retroviral Packaging Cell Line) forproducing Retrovirus were cultured in DMEM supplemented with 15% FBS,100 U ml-1 penicillin, 100 μg ml-1 streptomycin, sodium pyruvate (1 mM)and L-glutamine (4 mM) at 37° C. with 5% CO₂. Mouse embryonicfibroblasts (MEF) were cultured in DMEM supplemented with 15% FBS, 100 Uml-1 penicillin, 100 μg ml-1 streptomycin, sodium pyruvate (1 mM),L-glutamine (4 mM), L-ascorbic acid (50 uM) at 37° C. with low oxygen(4.5% O₂). iPSCs were derived in DMEM supplemented with 15% FBS, 100 Uml-1 penicillin, 100 μg ml-1 streptomycin, sodium pyruvate (1 mM),L-glutamine (4 mM), 1000 U/ml LIF, 0.1 mM 2-mercaptoethanol, and 50 μgml-1 ascorbic acid at 37° C. with 5% CO₂ and 4.5% O₂. iPSCs forblastocyst injection were cultured on feeders in DMEM supplemented with13% Knockout Serum Replacement (Gibco™), 2% FBS, 100 U ml-1 penicillin,100 μg ml-1 streptomycin, sodium pyruvate (1 mM), L-glutamine (4 mM),L-ascorbic acid (50 uM), 1000 U/ml LIF, beta mercaptoethanol, MEKinhibitor (104) and GSK3 inhibitor (304) at 37° C. with 5% CO₂.Conventional reprogramming media consisted of DMEM supplemented with 15%FBS, 100 U ml-1 penicillin, 100 μg ml-1 streptomycin, sodium pyruvate (1mM), L-glutamine (4 mM), 1000 U/ml LIF, 0.1 mM 2-mercaptoethanol unlessotherwise noted. For some experiments, media was supplemented with MEKinhibitor (104), GSK3 inhibitor (304), Dot11 inhibitor (1 uM) orascorbate (50 ug/ml).

Primary MEFs were derived from E13.5 embryos isolated from intercrossesbetween mice homozygous for Col1a1::tetOP-OKSM; Oct4-GFP and Rosa26M2rtTA, respectively. Embryos were dissected carefully excludinginternal organs, heads, limbs and tails and only carcasses were used forMEF derivation. Tissues were chopped into small clumps using scalpels,trypsinized and cultured in MEF medium at low O₂ (4%). MEFs were frozenat passage 0 upon derivation and used at passage 1 for all downstreamtransduction and reprogramming experiments. All MEFs were cultured atlow O₂ (4%) and supplemented with ascorbate to prevent replicativesenescence before OKSM overexpression.

Reprogramming experiments were initiated at low oxygen levels during doxinduction and completed at normal oxygen levels (20%) for mir-Eexperiments. Mir-30 assays were performed continuously at normal oxygenlevels. HSPCs were isolated from fetal livers of the same mid-gestationreprogrammable transgenic embryos used for MEFs derivation, dissociatedby vigorous pipetting with a 1 ml tip, filtered using 35 μm nylon mesh,followed by red blood cell lysis and cultured in RPMI/FBS mediasupplemented with stem cell factor (SCF), I13 and I16 cytokines andtransduced as indicated in the schematic (FIG. 13A).

Arrayed shRNA Library and Screen

Single shRNA clones were picked from the master library at CSHL, arrayedin 12×96 well plates and sequence-verified individually using miR30backbone primers. An additional 200 unmatched clones were re-picked andsequenced to allow maximum coverage of the library. Double transgenicreprogrammable MEFs carrying the OKSM inducible cassette andconstitutive rtTA (Col1a1::tetOP-OKSM; R26-M2rtTA) were seeded at 10e4cells per well in 96 well plates in duplicates and infected with thecorresponding retroviral virus particles freshly produced and filteredin 96 well format.

48 hrs post transduction, MEFs from each row were trypsinized andtransposed into 6 well dished coated with 0.2% gelatin in standardreprogramming media supplemented with dox and G418 at 0.2 mg/ml for thefirst six days of OKSM expression. Dox was withdrawn at day 12, allowingstable iPSCs to form. iPSC colonies were then stained for alkalinephosphatase expression using Vector Red™ Alkaline Phosphatase SubstrateKit (VectorLabs™) according to the supplier's protocol. Plates werescanned with a Perfection V500 Photo scanner (Epson™) and automatedcounting was performed using a proprietary image-processing algorithm byNikon™. Reprogramming efficiency was calculated based on infectionefficiency and normalized to infections with control shRNAs targetingthe luciferase Renilla transcript.

Retrovirus Production, Transduction of MEFs and Derivation of iPSCs

Retroviral constructs were introduced into Platinum-E™ RetroviralPackaging cells using calcium phosphate transfection or lipofection aspreviously described (Zuber, J. et al. (2011) Nature Biotechnology29:79-83). shRNAs were transduced into primary MEFs carrying theCol1a1::tetOP-OKSM and R26-M2rtTA alleles as well as a Pou5f1-EGFPreporter at single copies. For transduction, 180,000 cells were platedper well of a 6-well dish; all vectors were transduced in biologicaltriplicate. After 36 hrs, transduced cells were selected with 0.5 mgml-1 G418 for 3 days and 0.25 mg ml-1 G418 for an additional 3 days. 3days after shRNA transduction, infected cells were washed with PBS (1×)and trypsinized with Trypsin-EDTA (1×) and 20,000 cells were plated intoa 6-well. OSKM expression was induced for 7 days and cells were culturedin DMEM supplemented with 15% FBS, 100 U ml-1 penicillin, 100 μg ml-1streptomycin, sodium pyruvate (1 mM), L-glutamine (4 mM), 1000 U/ml LIF,0.1 mM 2-Mercaptoethanol, 50 μg ml-1 sodium ascorbate and 1 μg ml-1doxycycline at 37° C. with lox oxygen (4.5% O₂) and 5% CO₂. After 7 daysof OSKM expression, cells were cultured for an additional 4 days withoutdoxycycline to withdraw OSKM transgene expression at 37° C. with 5% CO₂,ambient oxygen. Following trypsinization, cells were analyzed forOct4-GFP expression using a FACS BD LSRFortessa™ (BD Biosciences™), datawere analyzed using FlowJo™.

Phenotypic Characterization of iPSCs

Alkaline phosphatase activity was measured using an enzymatic assay foralkaline phosphatase (VECTOR Red™ Alkaline Phosphatase (AP) SubstrateKit) according to the manufacturer's protocol.

Flow Cytometry Analysis of Reprogramming Intermediates

Reprogramming intermediates and Pecam stains were performed aspreviously described⁶. All samples were analyzed on a MACSQuant™fluorescence cytometer (Miltenyi™).

Transdifferentiation Assays

Induced neurons were generated as described in the experimental scheme(FIG. 13D). CAF-1 or Renilla RNAi inducible transgenic MEFs weretransduced with Ascl1-inducible lentivirus, exposed to dox 24 hrs postinduction, cultured in MEF media for the first 48 hrs and switched toserum-free neuronal media (N3B27) supplemented with dox for anadditional 11 days. Cultures were fixed and stained for MAP2 aspreviously described³³. Pre-B cells (C10 line) were cultured in RPMIMedium, 10% charcoal stripped FBS (Invitrogen™), 2 mM L-Glutamine, 100unit/ml penicillin, 1000 ug/ml streptomycin), 55 uMbeta-mercaptoethanol. Pre-B cells were transduced 48 hrs beforeinitiating macrophage transdifferentiation with estradiol (E2). Fortransdifferentiation assays, cultures were transduced with lentiviralpLKO vectors obtained from the Broad Institute's RNAi consortium (emptyvector “null control” or vector carrying stem-loop shRNAs targetingChaf1a and Chaf1b subunits). Following selection of transduced cellswith puromycin, cells were seeded at le6 cells/ml and supplemented withE2 and macrophage cytokines (IL3 and CSF) as previously described³⁴. Alltime points were analyzed for Cd14 and Mac1 expression by flow cytometryon the same day.

Quantitative RT-PCR

RNA was extracted (Qiagen RNeasy™ mini kit) and reverse transcribed (GEIllustra™ ready-to-go RT-PCR beads) according to the supplier'sinstruction. Quantitative PCR was performed using SybrGreen™ mix and aBIO-RAD™ CFX connect cycler. Primers used were:

b-Act-F GCTGTATTCCCCTCCATCGTG b-Act-R CACGGTTGGCCTTAGGGTTCAG Chaf1b-RGGCTCCTTGCTGTCATTCATCTTCCAC Chaf1b-F CACCGCCGTCAGGATCTGGAAGTTGG Chaf1a-RGTGTCTTCCTCAACTTTCTCCTTGG Chaf1a-F CGCGGACAGCCGCGGCCGTGGATTGC.

SDS-PAGE and Western Blot Analysis

Whole-cell lysates from reprogramming intermediates were run on 4-20%gradient SDS-polyacrylamide gels and transferred to nitrocellulosemembrane (Bio-Rad™) by standard methods. Membranes were blocked for 1 hin 5% non-fat dry milk in 1×TBS with 0.05% Tween-20 (TBST), rinsed, andincubated with primary antibody diluted in 3% BSA in TBST overnight at4° C. The following primary antibodies were used: anti-Chaf1a (sc-10206,Santa Cruz™), anti-Chaf1b (sc-393662, Santa Cruz™), anti-TBP (ab818,Abcam™), HRP conjugate anti actin (AC-15, Sigma™) Blots were washed inTBST, incubated with HRP-conjugated secondary antibodies forsemi-quantitative Western blot analysis and IR dye 800CW or IR dye 680RDfor quantitative westerns, as indicated. Secondary antibodies for bothmethods were incubated in 5% milk in TBST for 1 hour at room temperature(except for anti-β-ACTIN-Peroxidase antibody, which was incubated for 15min), and washed again. HRP signal was detected by EnhancedChemiLuminescence™ (Perkin Elmer™) Fluorescent infrared signal wasdetected using LI-COR Odyssey™ imaging system.

ATAC-Seq Chromatin Assay

To generate ATAC-seq libraries, 50,000 cells were used and librarieswere constructed as previously described³⁹. Briefly, cells were washedin PBS twice, counted and nuclei were isolated from 100,000 cells using100 ul hypotonic buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1%NP40) to generate two independent transposition reactions. Nuclei weresplit in half and treated with 2.5 μL Tn5 Transposase (Illumina™) for 30min at 37° C. DNA from transposed nuclei was then isolated andPCR-amplified using barcoded Nextera™ primers (Illumina™). Library QCwas carried out using high sensitivity DNA bioanalyzer assay and qubitmeasurement and sequenced using paired end sequencing (PESO) onIllumina™ Hi-Seq 2500 platform.

Sono-Seq and ChIP-Seq Chromatin Assays

For all ChIP experiments, 10e7 reprogramming intermediates werecollected per library. Chromatin precipitation assays were performed aspreviously described (Bernstein, B. et al. (2005) Cell 120:169-181)using goat polyclonal anti-Sox2 antibody (AF2018, R&D™). Briefly, cellswere cross-linked on plate in 1% methanol-free formaldehyde andsnap-frozen in liquid nitrogen until processed.

Nuclei were isolated using 1 ml of cell lysis buffer (20 mM Tris pH8, 85mM KCL, 0.5% NP40 and 1×HALT protease inhibitor cocktail), resuspendedin nuclear lysis buffer (10 mM Tris-HCL pH7.5, 1% NP40, 0.5% Nadeoxycholate, 0.1% SDS, 1×HALT protease inhibitor cocktail) andsonicated using optimized pulses of a Branson sonifier (1 min ON/OFFpulses for 5 cycles) for ChIP-seq libraries and S220 Covaris™ sonicator(Settings: duty cycle 5%, intensity 6, cycles/burst 200, pulse length 60s, 20 cycles, 8° C.) for sono-seq input preparations. Sonications wereverified for both methods using the 2100 Bioanalyzer™.Immunoprecipitations were carried out by first adjusting saltconcentration in sheared chromatin to 167 mM NaCl and adding antibodies(bug of Sox2 antibody) and incubated for 3-4 hrs at 4 C. 50 μl Protein GDynabeads (Invitrogen™) were prepared for each IP reaction by washing2-3 times in ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 167 mM NaCl,0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA) and added for an additionalhour to pull down bound chromatin. Bead complexes were then washed 6times in RIPA buffer (20 mM Tris-HCL pH8.1, 1 mM EDTA, 140 mM NaCl, 1%Triton X-100, 0.1% SDS, 0.1% Na deoxycholate), then two times with RIPAwith high salt concentration (500 mM), then 2 washes in LiCL buffer (10mM tris-HCL pH 8.1, 1 mM EDTA, 1% DOC, 1% NP40, 250 mM LiCL) and 2 finalwashes in TE buffer. Complexes were then eluted and reverse cross-linkedin 50 ul ChIP elution buffer (10 mM Tris-HCL pH8, 5 mM EDTA, 300 mMNaCl, 01% SDS) and 8 ul of reverse crosslinking buffer (250 mM Tris-HClpH 6.5, 1.25M NaCl, 62.5 mM EDTA, 5 mg/mL Proteinase K, 62.5 ug/mL RNAseA) by incubation at 65° C. for 6 h. DNA was isolated using Ampure™ SPRIbeads and yield quantified using Qubit™ fluorometer. ChIP-seq librarieswere constructed from 10 ng of immunoprecipitated DNA using the NEBNext™ChIP-Seq Library Prep Reagent Set for Illumina™ (New England Biolabs™),following the supplier's protocol. Briefly, purified DNA wasend-repaired and dA-tailed. Following subsequent ligation of sequencingadaptors, ligated DNA was size-selected to isolate fragments in therange of 300-550 bp in length using Egels. Adaptor-ligated fragmentswere enriched in a 14-cycle PCR using Illumina™ multiplexing primers.Libraries were purified, analyzed for correct size distribution usingdsDNA High Sensitivity Chips on a 2100 Bioanalyzer™ (Agilent™), pooledand submitted for single-end 50 bp Illumina™ GAII high-throughputsequencing.

Sono-Seq Bioinformatics Analysis

The reads were aligned to mm9 using Bowtie with a unique mapping option(Langmead et al. (2009) Genome Biology 10:R25). The smoothed tag densityprofiles were generated using get.smoothed.tag.density function of theSPP R package with a 100-bp Gaussian kernel, 50-bp step and library sizenormalization (Kharchenko, P et al. (2008) Nature Biotechnology26:1351-1359). The positions of promoters and enhancers in ESCs and MEFswere obtained from publicly available data set³⁸. To access thesignificance of the difference in the enrichment values between CAF-1and Renilla knockdown samples, a paired Wilcoxon rank sum test was used.

ATAC-Seq Bioinformatics Analysis

The reads were aligned to the mouse genome mm9 using BWA version 0.7.8with −q 5-1 32-k 2 and paired option (Li and Durbin. (2009)Bioinformatics 25:1754-1760). Non-primary mapping, failed QC, duplicatesand non-paired reads were filtered. Reads from different chromosomes andchrM were also filtered. Only uniquely mapped reads were used for theanalysis. The read density profiles were generated using 150 bp windowwith a 20 bp step and were normalized by the library size. For thecomparison between Chaf1a.166 mutant and Renilla mutant, the readdensity profiles were further normalized using the mean values of allannotated promoters from mm9. The positions of promoters and enhancersin mESC and MEF were obtained from publicly available data sets inSono-seq analyses³⁸. The coordinates of the metagene plot insuper-enhancers were used from recently published datasets⁴². For eachsuperenhancer region, the tag density signals were averaged into 101bins, with the margin of 5 kb outside of super-enhancers. Significantlyenriched regions were detected using Hotspot with FDR=0.01 74. Thedifferential sites between CAF-1 KD and Renilla KD were identified usingDiffBind with p=0.05 for the consensus ATAC-seq peaks (Ross-Innes et al(2012) Nature 481:389-393). DiffBind uses statistical routines developedin edgeR (Robinson, M D et al. (2010) Bioinformatics 26:139-140. Aone-sided paired Wilcoxon rank sum test was used for the comparison inthe enrichment values between CAF-1 KD and Renilla KD.

Sox2 ChIP-Seq Bioinformatics Analysis

The reads were aligned to mm9 using Bowtie with a unique mapping option(Langmead et al. (2009) Genome Biology 10:R25). The smoothed tag densityprofiles for Sox2 were generated using get.smoothed.tag.density functionof the SPP R package with a 100-bp window, 50-bp step and library sizenormalization as in Sono-seq analyses. The log 2 fold enrichmentprofiles were generated using get.smoothed.enrichment.mle in SPP Rpackage. The same coordinates of promoters and enhancers in mESC and MEFwere used as in Sono-seq and ATAC-seq analyses. A paired Wilcoxon ranksum test was used for the comparison in the enrichment values betweenCAF-1 KD and Renilla KD for Sox2. For the peak comparison in Sox2between CAF-1 KD and Renilla KD, first the reads were subsampled to makethe sequencing depth the same from each condition as the number of peakscalled tends to increase as sequencing depth is deeper. Thesignificantly enriched peaks compared inputs were detected using SPPfind.binding.positions function with e-value+10 (Kharchenko, P et al.(2008), supra). The overlapped peaks were compared with a margin of 200bp distance. For the unique peaks, peaks which were present only fromone condition (CAF-1 KD or Renilla KD) were first identified. For thepeaks which were present from one condition, the enrichment values(input-subtracted tag counts) were compared between CAF-1 KD and RenillaKD. If the ratios between the enrichment values between conditionswere >2 fold, the peaks were considered as “unique” for one of theconditions. mESC Sox2 data was used from publicly available data sets⁴³and analyzed as described above.

H3K9Me3 ChIP-Seq Bioinformatics Analyses

ChIP sequencing data was mapped to the mouse genome (mm9) with Bowtie0.12.7 allowing up to three mismatches, and retaining uniquely mappingreads. To assess H3K9me3 signal distribution genome-wide, we divided thegenome in 5 Kb intervals, and for each interval, the ratio of RPMnormalized signal in the IP and input samples was calculated. Intervalswith less than 10 reads in the input samples (˜10% of all) were excludedfrom further analyses due to low coverage. Intervals overlappingspecific regions were extracted using the bedtools suite (Quinlan et al.(2010) Bioinformatics 26:841-842. RRR region annotations were obtainedfrom Matoba et al⁴⁵, and signal across all included 5 Kb intervals wasaveraged.

For H3K9me3 enrichment over TE bodies, the mm10 genome version was used,as this release contains the most recent TE annotations. The genomicregions corresponding to TE families annotated in the mm10 RepeatMaskertrack in the UCSC genome browser were extracted, and the normalized readcounts in IP to input samples were calculated for each family. Due tothe repetitive nature of TEs, all results were further validatedconsidering reads that map to multiple (up to 10000) positions in thegenome, and scaling read counts by the number of valid alignments. Thisthreshold for multiple mapping positions was chosen as it was previouslyshown to approximate results obtained allowing unlimited mappingpositions, but at a significantly improved computation speed (Pezic etal. (2014) Genes & Development 28:1410-1428). In all analyses, signalestimates based on uniquely mapping reads, and based on reads mapping tomultiple genomic positions, produced similar results.

RNA-Seq Analysis of Genes and Transposable Element BioinformaticsAnalysis

RNA sequencing data was first pre-processed using Reaper (Davis, M P etal. (2013) Methods 63:41-49) to remove any Illumina adapter sequencesand computationally depleted of ribosomal RNA sequences (GenBankidentifiers: 18S, NR_003278.3; 28S, NR_003279.1; 5S, D14832.1; and 5.8S,K01367.1) using Bowtie 0.12.7 allowing 3 mismatches 71. Forprotein-coding gene expression analyses, pre-processed data was mappedto the mouse genome (mm10) using Bowtie 0.12.7 71 allowing 3 mismatches,and retaining uniquely mapping reads. Mouse transcript annotations wereobtained from RefSeq, and reads corresponding to the exonic regions ofeach gene were calculated using a custom phyton script. For overlappinggenes, reads corresponding to overlapping regions were divided equally.Gene differential expression was analysed using the DESeq R package80.

For TE expression analyses, data was mapped to the mm10 genome with 0mismatches and considering reads that map to up to 10000 genomicpositions as in ChIP sequencing analyses. The number of readscorresponding to TE regions annotated by the UCSC RepeatMasker trackwere calculated, scaling by the number of valid alignments for eachread. Scaled reads for each TE family were summed, and normalized asRPM. Heatmaps were generated using the gplots R package, anddifferential expression analyses were performed using the DESeq Rpackage (Anders and Huber. (2010) Genome Biology 11:R106). Comparisonsof RNA-sequencing results from analyses based on uniquely mapping reads,and based on reads mapping to multiple genomic positions, showed verysimilar results.

Statistical Analyses

Unpaired student t test was used for statistical analysis in replicatesof cell biology experiments. All error bars represent means±SEM or STDEVof independent biological replicates as indicated. A probability valueof p<0.05 was considered statistically significant. Numbers of replicateexperiments (n) are shown in figure legends. All graphs with no errorbars represent n=1. To assess significant differences in signalenrichment at ESC promoters, enhancers or super-enhancers by Sono-seq,ATAC-seq and ChIP-seq analysis upon CAF-1 knockdown or Renillaknockdown, a paired Wilcoxon rank sum test was used, where it is assumedthat populations do not follow normal distributions. To identifydifferential ATAC-seq peaks between CAF-1 and Renilla knockdown samples,negative binomial models were used.

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1. A method of inducing differentiation of a cancer cell or cancer stem cell in vivo, the method comprising: administering an inhibitor of the CAF-1 complex to a subject having, or suspected of having cancer, thereby inducing differentiation of the cancer cell or cancer stem cell in vivo.
 2. The method of claim 1, wherein the cancer comprises leukemia.
 3. The method of claim 1, wherein the inhibitor comprises an RNA interference molecule or an antibody.
 4. The method of claim 3, wherein the RNA interference molecule comprises an siRNA or an shRNA. 